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Rational Approach to Design of Melanocortin Ligands and Receptor Chimeras

Permanent Link: http://ufdc.ufl.edu/UFE0021902/00001

Material Information

Title: Rational Approach to Design of Melanocortin Ligands and Receptor Chimeras
Physical Description: 1 online resource (334 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: benzodiazepine, cation, chimera, mc2r, mc4r, melanocortin, mt2, obesity, pi, shu9119
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The melanocortin system is made up of five G protein-coupled receptors (GPCRs), the endogenous agonists ?-MSH, ?-MSH, ?1-MSH, ?2-MSH, and adrenocorticotropic hormone (ACTH), and the only two known antagonists of GPCRs, agouti and agouti-related protein (AGRP). Many physiological processes have been linked to the melanocortin system, such as skin pigmentation, energy and weight homeostasis, and steroidogenesis. Obesity (BMI > 30) is a condition increasing in morbidity across the United States, and which may lead to serious complications such as type II diabetes milletus, hypertension, stroke, heart disease, and cancer. It has been shown through targeted disruption of the MC4R that this receptor plays a role in the development of obesity. Structure-activity relationship studies of the endogenous agonist ?-MSH have identified key amino acids involved in ligand binding to the receptor and have aided in the design of synthetic ligands more potent than ?-MSH at the MC4R. It is hypothesized that potent agonists selective for the MC4R may be used as therapeutic agents to treat obesity. This dissertation investigates three experimental methods used to provide information about putative ligand-receptor interactions for the design of drugs targeting the melanocortin system: Structure-Activity Relationship Studies of Peptides Design of Peptidomimetics and Small Molecules Receptor Mutagenesis and Chimeric Receptors The first section reports the synthesis and evaluation of derivatives of the melanocortin synthetic ligands MTII and SHU9119. It is hypothesized that there may be a cation-? interaction between an Arg and the adjacent aromatic amino acid which may be involved in agonist and antagonist activity. To investigate this theory, a series of peptides was synthesized which incorporate six aromatic amino acids: DPhe, Phe, DNal(2?), Nal(2?), DNal(1?) and Nal(1?) coupled with either Arg, Lys, or Ala. A cation-? interaction is indicated by the potency series Arg > Lys > Ala. The second section describes the synthesis of a series of 1,4-benzodiazepine-2,5-diones as small molecule ligands of the melanocortin receptors. These compounds were designed based on the core tetrapeptide sequence His-Phe-Arg-Trp present in all melanocortin agonists. Compounds were synthesized with significant agonist activity at all receptors. The final section involves the construction and characterization of a series of hMC2R (human Melanocortin-2 Receptor) and hMC4R (human Melanocortin-4 Receptor) chimeras in order to clarify the unique properties of the hMC2R. It is hypothesized that the MC2R and ACTH may be involved in the homeostasis of human hypertension, and therefore correlated to heart disease and stroke. The study proposed herein attempts to understand the molecular mechanism of how ACTH stimulates the MC2R. The results of these studies illustrate important intermolecular interactions present in melanocortin ligands which may be used in the future to design more potent and bioavailable drugs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Haskell-Luevano, Carrie.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021902:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021902/00001

Material Information

Title: Rational Approach to Design of Melanocortin Ligands and Receptor Chimeras
Physical Description: 1 online resource (334 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: benzodiazepine, cation, chimera, mc2r, mc4r, melanocortin, mt2, obesity, pi, shu9119
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The melanocortin system is made up of five G protein-coupled receptors (GPCRs), the endogenous agonists ?-MSH, ?-MSH, ?1-MSH, ?2-MSH, and adrenocorticotropic hormone (ACTH), and the only two known antagonists of GPCRs, agouti and agouti-related protein (AGRP). Many physiological processes have been linked to the melanocortin system, such as skin pigmentation, energy and weight homeostasis, and steroidogenesis. Obesity (BMI > 30) is a condition increasing in morbidity across the United States, and which may lead to serious complications such as type II diabetes milletus, hypertension, stroke, heart disease, and cancer. It has been shown through targeted disruption of the MC4R that this receptor plays a role in the development of obesity. Structure-activity relationship studies of the endogenous agonist ?-MSH have identified key amino acids involved in ligand binding to the receptor and have aided in the design of synthetic ligands more potent than ?-MSH at the MC4R. It is hypothesized that potent agonists selective for the MC4R may be used as therapeutic agents to treat obesity. This dissertation investigates three experimental methods used to provide information about putative ligand-receptor interactions for the design of drugs targeting the melanocortin system: Structure-Activity Relationship Studies of Peptides Design of Peptidomimetics and Small Molecules Receptor Mutagenesis and Chimeric Receptors The first section reports the synthesis and evaluation of derivatives of the melanocortin synthetic ligands MTII and SHU9119. It is hypothesized that there may be a cation-? interaction between an Arg and the adjacent aromatic amino acid which may be involved in agonist and antagonist activity. To investigate this theory, a series of peptides was synthesized which incorporate six aromatic amino acids: DPhe, Phe, DNal(2?), Nal(2?), DNal(1?) and Nal(1?) coupled with either Arg, Lys, or Ala. A cation-? interaction is indicated by the potency series Arg > Lys > Ala. The second section describes the synthesis of a series of 1,4-benzodiazepine-2,5-diones as small molecule ligands of the melanocortin receptors. These compounds were designed based on the core tetrapeptide sequence His-Phe-Arg-Trp present in all melanocortin agonists. Compounds were synthesized with significant agonist activity at all receptors. The final section involves the construction and characterization of a series of hMC2R (human Melanocortin-2 Receptor) and hMC4R (human Melanocortin-4 Receptor) chimeras in order to clarify the unique properties of the hMC2R. It is hypothesized that the MC2R and ACTH may be involved in the homeostasis of human hypertension, and therefore correlated to heart disease and stroke. The study proposed herein attempts to understand the molecular mechanism of how ACTH stimulates the MC2R. The results of these studies illustrate important intermolecular interactions present in melanocortin ligands which may be used in the future to design more potent and bioavailable drugs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Haskell-Luevano, Carrie.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021902:00001


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1 RATIONAL APPROACH TO DESIGN OF M ELANOCORTIN LIGANDS AND RECEPTOR CHIMERAS By KRISTA RENNER WILSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Krista Renner Wilson

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3 To everyone who prayed me through my education.

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4 ACKNOWLEDGMENTS I would like to thank a ll who have been instrumental in my life and education. I especially thank my parents who have always been there to love and support me. Without their help I would not be who I am today. I also thank all the pa st and present members of the Haskell-Luevano laboratory for teaching me everything I know about chemistry and molecular biology. I especially thank Dr. Andrzej Wilczynski and Dr Christine Joseph for teaching me peptide and organic synthesis and Dr. Bettina Proneth and Dr. Zhimin Xiang for teaching me molecular biology and pharmacology skills. I also thank all the technicians who were involved in assaying my compounds over the years; th eir contribution was essential to my success. Special thanks go to Jay Schaub for his help in proofreading th is dissertation and help ing with the list of abbreviations. I would like to thank my superv isory committee members; especially Dr. Carrie Haskell-Luevano, for her support over the years. I also thank Dr. Kenne th Sloan, Dr. Hendrik Luesch and Dr. Sally Litherland fo r their counsel. I would also lik e to thank Dr. Margaret James and Dr. Raymond Bergeron for their role in my success. Finally I would like to thank my husba nd, Brandon Wilson. Without his love and encouragement I would never have made it this far. I thank him for driving me to the lab in the middle of the night to refill dry ice or check the HPLC and helping me label countless tubes and plates. I thank God every day for bringing him into my life. I also acknowledge the University of Flor ida Alumni Association for supporting my education for the last 4 years and the NIH for providing grants that supported my research.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............24 CHAPTER 1 INTRODUCTION TO DRUG DISCOVERY........................................................................26 Rational Drug Design........................................................................................................... ..26 Structure-Activity Relations hip Studies of Peptides..............................................................27 Design of Peptidomimetics and Small Molecules..................................................................32 Secondary Structural Features of Peptides......................................................................32 Peptide Modification in the Design of Peptidomimetics.................................................37 Global constraints.....................................................................................................38 Local constraints......................................................................................................41 Design of Small Molecules.............................................................................................43 Receptor Mutagenesis and Chimeric Receptors.....................................................................45 2 THE MELANOCORTIN SYSTEM.......................................................................................47 The Melanocortin Receptors...................................................................................................47 The Melanocortin Ligands......................................................................................................5 0 Diseases Associated with the Melanocortin System..............................................................57 3 GENERAL METHODOLOGIES 1: CHEMISTRY..............................................................61 Solid Phase Peptide Synthesis................................................................................................61 Merrifield Approach........................................................................................................61 The Fmoc Synthetic Strategy..........................................................................................64 Resins for SPPS........................................................................................................65 Side chain protecting groups....................................................................................68 Coupling methods....................................................................................................70 Colorometric monitoring methods...........................................................................76 Solid Phase 1,4-Benzodiazepine-2,5-dione Synthesis............................................................77 Solid Phase Organic Synthesis........................................................................................78 Colorimetric Monitoring.................................................................................................84 Experimental Details........................................................................................................... ...85 Synthesis of MTII-based CationPeptides....................................................................85 1,4-Benzodiazepine-2,5-diones.......................................................................................87 Ligand Purification and Analysis....................................................................................90

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6 Reporter Gene Assay: -Galactosidase...........................................................................90 4 GENERAL METHODOLOGIES 2: MOLECULAR BIOLOGY.........................................91 Molecular Cloning.............................................................................................................. ....91 Structure of DNA............................................................................................................91 Replication of DNA.................................................................................................93 Transcription of DNA..............................................................................................97 Translation into protein............................................................................................98 The PCR reaction...................................................................................................101 Restriction endonuclease digestion........................................................................103 Gel electrophoresis.................................................................................................104 Sequencing.............................................................................................................106 Bacterial Expression Vectors........................................................................................107 Cell Culture and Transfection...............................................................................................111 Mammalian Cell Lines..................................................................................................111 Transfection................................................................................................................... 112 Functional Assay............................................................................................................... ...113 Pharmacology Definitions.............................................................................................113 Luciferase Assay...........................................................................................................115 Functional Assay: cAMP Assay....................................................................................117 Experimental Methods..........................................................................................................1 18 Cell Culture and Transfection.......................................................................................118 Site-Directed Receptor Mutagenesis.............................................................................119 Real Time Polymerase Chain Reaction (RT-PCR).......................................................122 Fluorescence-Activated Cell Sorting (FACS)...............................................................124 Luciferase Reporter Gene Assay...................................................................................125 Functional Assay: cAMP Assay....................................................................................125 Data Analysis.................................................................................................................1 26 5 STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF PEPTIDES: A STUDY OF INTRAMOLECULAR CATIONINTERACTIONS IN MELANOCORTIN AGONISTS AND THE EFFECTS ON AGONIST AND ANTAGONIST SELECTIVITY.................................................................................................................... .127 Introduction................................................................................................................... ........127 Background Pharmacology...........................................................................................133 Results........................................................................................................................ ...........137 Discussion..................................................................................................................... ........143 D-Phenylalanine............................................................................................................144 Phenylalanine................................................................................................................14 5 D-Napthylalanine (2’)...................................................................................................146 Napthylalanine (2’)........................................................................................................147 D-Napthylalanine (1’)...................................................................................................148 Napthylalanine (1’)........................................................................................................149 Conformation Effects....................................................................................................150 Receptor Selectivity.......................................................................................................150

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7 Agonist vs. Antagonist Selectivity................................................................................152 Conclusions...................................................................................................................1 52 6 DESIGN OF PEPTIDOMIMETI CS AND SMALL MOLECULES: 1,4BENZODIAZEPINE-2,5-DIONES AS NM MELANOCORTIN AGONISTS..................153 Introduction................................................................................................................... ........153 Results........................................................................................................................ ...........166 Discussion..................................................................................................................... ........168 Receptor Specificities....................................................................................................179 Characterization Data.......................................................................................................... .180 7 CONSTRUCTION AND CHARACTERIZATION OF MELANOCORTIN-2 AND -4 RECEPTOR CHIMERAS....................................................................................................198 Introduction................................................................................................................... ........198 Results........................................................................................................................ ...........205 Flag-tagged hMC2R......................................................................................................205 Real-time PCR...............................................................................................................208 Chimera Construction....................................................................................................210 Fluorescence Activated Cell Sorting.............................................................................212 Luciferase Assay...........................................................................................................217 Functional Assay: cAMP...............................................................................................220 Discussion..................................................................................................................... ........221 Chimeras in HEK293 Cell Lines...................................................................................221 Chimeras in OS3 Cell Lines..........................................................................................228 Conclusions and Future Directions...............................................................................230 8 CONCLUDING REMARKS................................................................................................232 Structure-Activity Relationship Studies Of Peptides: A Study Of Intramolecular CationInteractions In Melanocortin Agoni sts And The Effects On Agonist And Antagonist Selectivity.......................................................................................................23 2 Design Of Peptidomimetics And Small Mol ecules: 1,4-Benzodiazepine-2,5-Diones As nM Melanocortin Agonists...............................................................................................233 Construction And Characterization Of Me lanocortin-2 And -4 Receptor Chimeras...........234 APPENDIX A 1H-NMR of 1,4-Benzodiazepine-2,5-Diones.......................................................................237 B DNA Sequences and Restriction Maps of Chimeras............................................................258 C Histograms of FACS Chimera Data.....................................................................................273 LIST OF REFERENCES............................................................................................................. 302 BIOGRAPHICAL SKETCH.......................................................................................................334

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8 LIST OF TABLES Table page 1-1 Secondary structural features induced by common amino acids.......................................35 1-2 Methods of disulfide and lactam bridge formation............................................................38 2-1 Ligand potency series for the five melanocortin subtypes.................................................51 2-2 Amino acid sequences of the endogenous and synthetic melanocortin agonists...............51 2-3 Truncation study of ACTH tested at the mMC2R.............................................................54 3-1 Common types of resin used in SPPS................................................................................66 3-2 Side chain protecting groups commonly used in SPPS.....................................................69 3-3 Phosphonium and aminium-based coupling reagents........................................................75 4-1 Diagram of mRNA codons translated to amino acid.........................................................99 4-2 Comparison of luciferase and cAMP assays....................................................................118 5-1 SAR Studies of Selected Melanocortin Tetrapeptides and Pharmacology Data of Arg Substitutions.................................................................................................................. ...134 5-2 Antagonists of the MC3R a nd MC4R containing DNal(2’)............................................135 5-3 Analytical Data for Peptid es Synthesized in the CationStudy....................................138 5-4 Pharmacology Data for Peptides in the CationStudy..................................................139 6-1 Pharmacology data of compounds s ynthesized by Dr. Christine Joseph.........................163 6-2 Analytical data of 1,4-benzodiazepine2,5-diones synthesized in this study..................164 6-3 Pharmacology data of 1,4-benzodiazepin e-2,5-diones synthesized in this study by Krista Wilson.................................................................................................................. .165 7-1 Primers for insertion of Fl ag tag into hMC2R template..................................................206 7-2 Restriction enzyme recognition sites...............................................................................212 7-3 Primers for insertion of unique restric tion sites in hMC2R and hMC4R templates........212 7-4 Functional activity of -MSH and ACTH(1-24) at selected chimeras............................220

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9 LIST OF FIGURES Figure page 1-1 Flowchart of drug design from biologically active peptides.............................................26 1-2 Graphical description of Structure-Activity Relationship (SAR) studies of peptides.......28 1-3 L versus D forms of phenylalanine and effects on -MSH and NDP-MSH potency........30 1-4 Potential ligand-receptor inte ractions at the binding pocket..............................................31 1-5 Peptide torsion angles defining s econdary structures and conformation...........................32 1-6 Representative Ramachandran plot depicting torsion angles defining common peptide secondary stru ctural features.................................................................................33 1-7 Common secondary structural features of peptides...........................................................35 1-8 Reverse turn structures found in peptides..........................................................................37 1-9 Disulfide Cyclization of Peptides......................................................................................39 1-10 Lactam bridge cyclization of peptides...............................................................................40 1-11 Small molecules and peptomimetics incorporating and -turn mimetic structures.......41 1-12 Unnatural, constrained amino acids to control peptide conformation...............................42 1-13 Backbone modifications of peptides..................................................................................43 1-14 Privileged structures often used in small molecule design................................................44 2-1 The Melanocortin system...................................................................................................4 7 2-2 Posttranslational processing of PO MC into the melanocortin agonists.............................50 2-3 ACTH truncated to -MSH................................................................................................53 2-4 Partial sequences of the melanocortin antagonists hAGRP and hAgouti depicting the five disulfide bonds in each ligand....................................................................................55 3-1 General method of solid phase peptid e synthesis including deprotection, coupling and cleavage steps............................................................................................................. .62 3-2 General Boc chemistry synthesis strategy.........................................................................63 3-3 Proposed mechanism for acidcatalyzed Boc deprotection...............................................63

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10 3-4 General Fmoc synthetic strategy........................................................................................64 3-5. Proposed mechanism for base -catalyzed Fmoc removal........................................................65 3-6 Diketopiperizine formation during sy nthesis using a hydroxymethyl linker and proline as the first amino acid............................................................................................67 3-7 Common side reactions observe d during amino acid activation.......................................70 3-8 Mechanism of amino acid coupling us ing carbodiimide coupling reagents......................71 3-9 Mechanism of amino acid coupling us ing carbodiimides through symmetrical anhydride formation...........................................................................................................7 2 3-10 Structures of common hydroxybe nzotriazoles used in coupling.......................................72 3-11 Proposed mechanism of amino acid coup ling through active ester formation using carbodiimides and HOBt as coupling reagents..................................................................73 3-12 Proposed mechanism of oxazolone forma tion leading to amino acid coupling when carbodiimides are used as coupling reagents.....................................................................73 3-13 Structures of phosphonium and aminium salts used in coupling reactions.......................74 3-14 N-terminal guanidation of peptide chai n when HBTU is the coupling reagent................74 3-15 Possible mechanism of primary amine detection using ninhydrin....................................77 3-16 Complex formed from the reaction of chloranil with a secondary amine.........................77 3-17 Structures of common benzodiazepine templates..............................................................78 3-18 Bunin et al synthesis of a 1,4-benzodiazepine-2-one.......................................................79 3-19 Boojamra et al synthesis of a 1,4-benzodiazepine-2,5-dione...........................................80 3-20 Keating et al synthesis of 1,4-benz odiazepine-2,5-diones................................................81 3-21 Bhalay et al synthesis of 1,4-benzodiazepine-2-ones.......................................................81 3-22 Mayer et al synthesis of 1,4-benzodiazepine-2-ones........................................................82 3-23 Lee et al synthesis of 1,5-be nzodiazepine-2-ones............................................................83 3-24 Zhang et al synthesis of 1,4-benzodiazepine-2-ones........................................................83 3-25 Reaction of an aldehyde with DNPH to give a red complex.............................................85 4-1 Structure of DNA and bases..............................................................................................91

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11 4-2 Formation of a phosphodiester bond during DNA synthesis.............................................92 4-3 Structure of the replication fork.........................................................................................9 4 4-4 DNA replication of circular and linear DNA.....................................................................95 4-5 The rolling circle method of DNA replication in prokaryotes...........................................96 4-6 Structures of DNA and RNA nucleotides..........................................................................97 4-7 Model of DNA transcription..............................................................................................98 4-8 Model of DNA translation...............................................................................................100 4-9 The PCR reaction........................................................................................................... ..102 4-10 Reagents for gel electrophoresis......................................................................................105 4-11 Model showing how BDT sequenc ing is read by the detector........................................107 4-12 Bacterial growth curve.................................................................................................... .108 4-13 Pharmacology curves....................................................................................................... 115 4-14 Luciferase reporter gene assay.........................................................................................116 5-1 Cationinteraction between the positive charge of arginine and the negative potential on the face of the Phe aromatic system.............................................................127 5-2 Structure of natural and unnatura l amino acids used in this study..................................128 5-3 Benzene.................................................................................................................... ........129 5-4 Cationinteractions of Arg and Lys with Phe and water...............................................131 5-5 Structure and pharmacology of the synt hetic melanocortin agonist MTII and the synthetic melanocortin antagonist SHU9119...................................................................132 5-6 Schematic of a cationinteraction between the cationi c side chain of Arg and the negative potential of the systems of DPhe and DNal(2’).............................................137 5-7 Bar graph of peptides 15, 21 and 22 with DPhe as the aromatic system at the mMC1,3-5R.....................................................................................................................1 45 5-8 Bar graph of peptides 16, 23 and 24 with Phe as the aromatic system at the mMC1,35R............................................................................................................................. ........146 5-9 Bar graph of peptides 17, 25 and 26 with DNal(2’) as the aromatic system at the mMC1,3-5R.....................................................................................................................1 47

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12 5-10 Bar graph of peptides 18, 27 and 28 with Nal(2’) as the aromatic system at the mMC1,3-5R.....................................................................................................................1 48 5-11 Bar graph of peptides 19, 29 and 30 with DNal(1’) as the aromatic system at the mMC1,3-5R.....................................................................................................................1 49 5-12 Bar graph of peptides 20, 31 and 32 with Nal(1’) as the aromatic system at the mMC1,3-5R.....................................................................................................................1 50 6-1 Structures of benzodiazepines..........................................................................................153 6-2 Small molecules with activity at the me lanocortin receptors and based on privileged structure templates...........................................................................................................1 55 6-3 Structures of 1,4-benzodiazepine-2,5-di ones synthesized in this study by KRW...........161 6-4 Structures of building blocks used to synthesize 1,4-benzodiazepine-2,5-diones 3958............................................................................................................................. .........162 6-5 Structure of Compound 37, a nanomolar agonist at all mela nocortin receptors tested..171 7-1 Amino acid alignment of huma n melanocortin receptors 1-5.........................................199 7-2 -MSH is formed from the e ndogenous truncation of ACTH.........................................200 7-3 Schematic of chimeras.....................................................................................................2 01 7-4 Addition of Flag tag to the hMC2R gene.........................................................................205 7-5 Characterization of HEK293 and OS3 cells stably expressing the Flag-hMC2R...........207 7-6 Gene expression assay of hMC2R-OS3, hMC4R-HEK, and OS3 cells..........................208 7-8 Schematic of chimera construction..................................................................................211 7-9 Diagram of unique restriction sites used during this study and th eir locations in the receptor....................................................................................................................... .....211 7-10 Schematic of FACS experiment.......................................................................................213 7-11 Summary of FACS data of wild type receptors and chimeras expressed in HEK293 cells.......................................................................................................................... ........214 7-12 Summary of FACS data of wild type receptors and chimeras expressed in OS3 cells...215 7-13 Histograms of FACS data from thr Flag -hMC4R construct expressed in both OS3 and HEK cell lines...........................................................................................................21 6

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13 7-14 Summary of functional data from the luciferase assay performed on hMC2R wild type receptors and chimeras expressed in HEK293 cells................................................218 7-15 Summary of functional data from the luciferase assay performed on hMC4R wild type receptors and chimeras expressed in HEK293 cells................................................219 7-16 Correlation plots of HEK hMC2R chimera data..............................................................224 7-17 Amino acid alignment of all mela nocortin receptors and chimeras................................227 7-18 Time course of HEK and OS3 cell li nes transiently tran sfected with GFP.....................229 A-1 Compound 39 (KRW9-70E) 1H-NMR, 3-(4-aminobutyl)-1-benzyl-3,4-dihydro-1Hbenzo[e][1,4]diazepine-2,5-dione. .................................................................................238 A-2 Compound 40 (KRW5-37) 1H-NMR, 3-Benzyl-1-biphenyl-2-ylmethyl-3,4dihydro-1H-benzo[e][1,4]diazepine-2,5-dione. ............................................................239 A-3 Compound 41 (KRW 9-70J) 1H-NMR, 1-(3-(1-(biphenyl-2-ylmethyl)-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4] diazepin-3-yl)propyl)urea. ................................240 A-4 Compound 42 (KRW9-70F) 1H-NMR, 3-(4-aminobutyl)-1-(biphenyl-2-ylmethyl)3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione. ......................................................241 A-5 Compound 43 (KRW9-70A) 1H-NMR, 3-benzyl-1-(naphthalen-1-ylmethyl)-3,4dihydro-1H-benzo[e][1,4]diazepine-2,5-dione. ............................................................242 A-6 Compound 44 (KRW9-70K) 1H-NMR, 1-(3-(1-(naphthalen-1-ylmethyl)-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4] diazepin-3-yl)propyl)urea. ................................243 A-7 Compound 45 (KRW9-70G) 1H-NMR, 3-(4-aminobutyl)-1-(naphthalen-1ylmethyl)-3,4-dihydro-1H-ben zo[e][1,4]diazepine-2,5-dione. ....................................244 A-8 Compound 46 (KRW4-91) 1H-NMR, 3-Benzyl-1-propyl-3,4-dihydro-1Hbenzo[e][1,4]diazepine-2,5-dione. .................................................................................245 A-9 Compound 47 (KRW5-25) 1H-NMR, 3-Benzyl-1-butyl-3,4-dihydro-1Hbenzo[e][1,4]diazepine-2,5-dione. .................................................................................246 A-10 Compound 48 (KRW9-70B) 1H-NMR, 1,3-dibenzyl-8-methyl-3,4-dihydro-1Hbenzo[e][1,4]diazepine-2,5-dione. .................................................................................247 A-11 Compound 49 (KRW9-70L) 1H-NMR, 1-(3-(1-benzyl-9-methyl-2,5-dioxo-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea. ............................................248 A-12 Compound 50 (KRW9-70H) 1H-NMR, 3-(4-aminobutyl)-1-benzyl-9-methyl-3,4dihydro-1H-benzo[e][1,4]diazepine-2,5-dione. ............................................................249

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14 A-13 Compound 51 (KRW9-70C) 1H-NMR, 1-(3-(1-benzyl-8-chloro-2,5-dioxo-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea. ............................................250 A-14 Compound 52 (KRW6-121A) 1H-NMR, N-[3-(1-Benzyl-8-chloro-2,5-dioxo-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-propyl]-guanidine. ................................251 A-15 Compound 53 (KRW9-70D) 1H-NMR, 3-benzyl-1-(biphenyl-2-ylmethyl)-8-chloro3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione .......................................................252 A-16 Compound 54 (KRW9-70M) 1H-NMR, 1-(3-(1-(biphenyl-2-ylmethyl)-8-chloro2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e ][1,4]diazepin-3-yl)propyl)urea ................253 A-17 Compound 55 (KRW9-70I) 1H-NMR, 3-(4-aminobutyl)-1-(biphenyl-2-ylmethyl)8-chloro-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione .......................................254 A-18 Compound 56 (KRW5-49) 1H-NMR, 3-Benzyl-8-chloro-1-naphthalen-2-ylmethyl3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione .......................................................255 A-19 Compound 57 (KRW9-70N) 1H-NMR, 1-(3-(8-chloro-1-(naphthalen-1-ylmethyl)2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e ][1,4]diazepin-3-yl)propyl)urea ................256 A-20 Compound 58 (KRW7-05A) 1H-NMR, 3-(4-Amino-butyl)-8-chloro-1-naphthalen2-ylmethyl-3,4-dihydro-1H-ben zo[e][1,4]diazepine-2,5-dione ...................................257 B-1 Flag-hMC2R DNA Sequence..........................................................................................259 B-2 Chimera 2C1 DNA Sequence..........................................................................................260 B-3 Chimera 2C2 DNA Sequence..........................................................................................261 B-5 Chimera 2C4 DNA Sequence..........................................................................................263 B-6 Chimera 2C5 DNA Sequence..........................................................................................264 B-7 Chimera 2C6 DNA Sequence..........................................................................................265 B-8 Flag-hMC4R DNA Sequence..........................................................................................266 B-9 Chimera 4C1 DNA Sequence..........................................................................................267 B-10 Chimera 4C2 DNA Sequence..........................................................................................268 B-11 Chimera 4C3 DNA Sequence..........................................................................................269 B-12 Chimera 4C4 DNA Sequence..........................................................................................270 B-13 Chimera 4C5 DNA Sequence..........................................................................................271 B-14 Chimera 4C6 DNA Sequence..........................................................................................272

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15 C-1 Flag-hMC2R in HEK cells FACS Histogram..................................................................274 C-2 Chimera 2C1 in HEK cells FACS Histogram..................................................................275 C-3 Chimera 2C2 in HEK cells FACS Histogram..................................................................276 C-4 Chimera 2C3 in HEK cells FACS Histogram..................................................................277 C-5 Chimera 2C4 in HEK cells FACS Histogram..................................................................278 C-6 Chimera 2C5 in HEK cells FACS Histogram..................................................................279 C-7 Chimera 2C6 in HEK cells FACS Histogram..................................................................280 C-8 Flag-hMC4R in HEK cells FACS Histogram..................................................................281 C-9 Chimera 4C1 in HEK cells FACS Histogram..................................................................282 C-10 Chimera 4C2 in HEK cells FACS Histogram..................................................................283 C-11 Chimera 4C3 in HEK cells FACS Histogram..................................................................284 C-12 Chimera 4C4 in HEK cells FACS Histogram..................................................................285 C-13 Chimera 4C5 in HEK cells FACS Histogram..................................................................286 C-14 Chimera 4C6 in HEK cells FACS Histogram..................................................................287 C-15 Flag-hMC2R in OS3 cells FACS Histogram...................................................................288 C-16 Chimera 2C1 in OS3 cells FACS Histogram...................................................................289 C-17 Chimera 2C2 in OS3 cells FACS Histogram...................................................................290 C-18 Chimera 2C3 in OS3 cells FACS Histogram...................................................................291 C-20 Chimera 2C5 in OS3 cells FACS Histogram...................................................................293 C-21 Chimera 2C6 in OS3 cells FACS Histogram...................................................................294 C-22 Flag-hMC4R in OS3 cells FACS Histogram...................................................................295 C-23 Chimera 4C1 in OS3 cells FACS Histogram...................................................................296 C-24 Chimera 4C2 in OS3 cells FACS Histogram...................................................................297 C-25 Chimera 4C3 in OS3 cells FACS Histogram...................................................................298 C-26 Chimera 4C4 in OS3 cells FACS Histogram...................................................................299

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16 C-27 Chimera 4C5 in OS3 cells FACS Histogram...................................................................300 C-28 Chimera 4C6 in OS3 cells FACS Histogram...................................................................301

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17 LIST OF ABBREVIATIONS 2-Cl-Z 2-Chlorobenzyloxycarbonyl 3-Bom 3-Benzyloxymethyl A Ala, Alanine AA Amino Acid Acm Acetomidomethyl AcOH Acetic Acid ACS American Chemical Society ACTH Adrenocorticotropic Hormone AGRP Agouti Related Protein Alloc Allyloxycarbonyl Anc Amino-2-Napthylcarboxylic Acid AOP (7-Azabenzotriazol-1-Yloxy)-Tris(Dimethylamino)Phosphonium Hexafluorophosphate APC Allophycocyanin ASP Agouti Signaling Peptide AT1/AT2 Angiotensin I/II Receptor Atc Amino-Tetrahydro-2-Napthylcarboxylic Acid ATP Adenosine Triphosphate BAL Backbone Amide Linker Resin BBB Blood Brain Barrier BMI Body Mass Index Boc Tert -Butyloxycarbonyl BOP Benzotriazol-1-Yl-N-Oxy-T ris(Dimethylamino)Phosphonium Hexafluorophosphate BSA Bovine Serum Albumin

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18 Bu3P Tributylphosphine Bzl Benzyl C Cys, Cysteine cAMP Cyclic 5’-Adenosine Monophosphate CCK Cholecystokinin Choranil 2,3,5,6-Tetrachlo ro-1,4-Benzoquinone CO2 Carbon Dioxide COF3 Cobalt (III) Fluoride CPE Carboxypeptidase E CRE cAMP Response Element CRH Corticotropin Releasing Hormone CRLR Calcitonin Receptor-Like Receptor CTR Calcitonin Receptor D Asp, Aspartic Acid DCC Dicyclohexylcarbodiimide DCM Dichloromethane DIC Diisopropylcarbodiimide DIEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMEM Dulbecco Modified Eagle's Media (Dul becco Modified Eagle's Minimal Essential Medium) DMF N,N-Dimethylformamide DMS Dimethylsulfide DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DNal(1’) D-1-Napthylalanine

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19 DNal(2’) D-2-Napthylalanine DNPH Dinitrophenylhydrazine Dpr Diaminoproprionic Acid E Glu, Glutamic Acid EDC 1-[3-(Dimethylamino)Propyl]-1 ’-Ethylcarbodiimide Hydrochloride Et3N Triethyl Amine F Phe, Phenylalanine FACS Fluorescence-Activated Cell Sorting Falp Fat Tissue-Specific Low Molecular Weight Protein FGD Familial Glucocorticoid Deficiency Fmoc 9-Fluorenylmethyloxycarbonyl For Formyl G Gly, Glycine GABA -Aminobutyric Acid GPCR G Protein Coupled Receptor H His, Histine HATU N-[(Dimethylamino)-1H-1,2,3-Triazolo [4,5]Pyridine-1-Ylmethylmethanaminium Hexafluorophosphate N-Oxide HBTU N-[1H-Benzotriazole-1-Yl)(Dimet hylamino)Methylamino)Methylene]-NMethylmethanaminium Hexafluorophosphate N-Oxide HEK293 Human Embryonic Kidney Cell Line 293 HF Hydrogen Fluoride Hg(II) Mercury (II) HIV Human Immunodeficiency Virus HOAt 1-Hydroxy-7-Azabenzotriazole HOBt 3-Hydroxybenzotriazole

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20 HPA Axis Hypothalamic-Pi tuitary-Adrenal Axis I Ile, Isoleucine IBMX Isobutylmethylxanthine ICV Intracerebroventricular IL-1 Interleukin 1 IL-10 Interleukin 10 IL-6 Interleukin 6 IP3 Inositol Triphosphate K Lys, Lysine KCl Potassium Chloride KO Knockout L Leu, Leucine M Met, Methionine MC1R Melanocortin-1 Receptor MC2R Melanocortin-2 Receptor MC3R Melanocortin-3 Receptor MC4R Melanocortin-4 Receptor MC5R Melanocortin-5 Receptor MCM Minichromosome Maintenance MCR Melanocortin Receptor MeOH Methanol MgCl2 Magnesium Chloride MRAP Melanocortin-2 Receptor Accessory Protein mRNA Messenger Ribonucleic Acid MsCl Mesyl Chloride

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21 MSH Melanocyte Stimulating Hormone MT-I Melanotan I MTII Melanotan II N Asn, Asparagine Na/NH3 Sodium/Ammonia NaBH(OAc)3 Sodium Triacetoxyborohydride NaH Sodium Hydride NaOH Sodium Hydroxide NaOtBu Sodium Tert -Butoxide NAT N-Acetyltransferase nBuLi N-Butyllithium NDP-MSH Norleucine, D-Phenylalanin e-Melanocyte Stimulating Hormone NHANES National Health And Nutrition Examination Survey Nle Norleucine nM Nanomolar NMP N-Methyl Pyrollidinone NMR Nuclear Magnetic Resonance Nup 50 Nucleopore 50 OAllyl Allyl Ester OD Optical Density ONPG Ortho -Nitrophenyl-Galactoside ORC Origin Recognition Complex Orn Ornithine OtBu Tert -Butyl Ester P Pro, Proline

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22 PAM Peptidyl -Amidating Monooxygenase Pbf 7-PentamethyldihydroBenzofuran-5-Sulfonyl PC1 Prohormone Convertase 1 PC2 Prohormone Convertase 2 Ph3PPd Palladium Triphenylphosphine PKC Protein Kinase C pM Picomolar POMC Proopiomelanocortin PyAOP (7-Azabenzotriazol-1-Yloxy)-Tris(Pyrrolidino)Phosphonium Hexafluorophosphate PyBOP Benzotriazol-1-Yl-N-Oxy-Tris(Py rrolidino)Phosphonium Hexafluorophosphate PyBrOP [Bromotris]-(Pyrrolidi no)Phosponium Hexafluorophosphate Q Gln, Glutamine R Arg, Arginine RAMP Receptor Activity Modifying Protein RP-HPLC Reverse Phase High Performance Liquid Chromatography rRNA Ribosomal RNA RT Room Temperature RT-PCR Real-Time Polymerase Chain Reaction S Ser, Serine SAR Structure Activity Relationship SEM Standard Error Of The Mean SnCl2 Tin (II) Chloride SPPS Solid Phase Peptide Synthesis StBu Tert -Butyl Thioether T Thr, Threonine

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23 TATU N-[(Dimethylamino)-1H-1,2,3-Tria zolo[4,5]Pyridine-1 -Ylmethylene]-NMethylmethanaminium Tetrafluoroborate N-Oxide TBTU N-[1H-Benzotriazole-1-Yl(Dimethyl amino)Methylene]-N-Methylmethanaminium Tetrafluoroborate N-Oxide tBu Tert -Butyl TFA Trifluoroacetic Acid THF Tetrahydrofuran Tic Amino-1,2,3,4-Tetrahydrois oquinoline-Carboxylic Acid TM Transmembrane TNF Tumor Necrosis Factor Alpha Tos Tosyl tRNA Transfer RNA Trt Trityl UV Ultraviolet V Val, Valine W Trp, Tryptophan Y Tyr, Tyrosine -MSH Alpha-Melanocyte Stimulating Hormone -MSH Beta-Melanocyte Stimulating Hormone -MSH Gamma-Melanocyt e Stimulating Hormone M Micromolar

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24 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RATIONAL APPROACH TO DESIGN OF M ELANOCORTIN LIGANDS AND RECEPTOR CHIMERAS By Krista Renner Wilson May 2008 Chair: Carrie Haskell-Luevano Major: Pharmaceutical Sciences The melanocortin system is made up of five G protein-coupled receptors (GPCRs), the endogenous agonists -MSH, -MSH, 1-MSH, 2-MSH, and adrenocorticotropic hormone (ACTH), and the only two known antagonists of GPCRs, agouti and agouti-related protein (AGRP). Many physiological processes have been linked to the melanocortin system, such as skin pigmentation, energy and weight homeostasis, and steroidogenesis. Obesity (BMI >30) is a condition increasing in morbidity across the United States, and which may lead to serious complications such as type II diabetes milletus, hypertension, stroke, heart disease, and cancer. It has been shown th rough targeted disruption of the MC4R that this receptor plays a role in the development of obes ity. Structure-activity relationship studies of the endogenous agonist -MSH have identified key amino acids involved in ligand binding to the receptor and have aided in the design of synthetic ligands more potent than -MSH at the MC4R. It is hypothesized that potent a gonists selective for th e MC4R may be used as therapeutic agents to treat obesity. This dissertation investigates three experimental methods used to provide information about putative ligand-receptor inte ractions for the design of dr ugs targeting the melanocortin system:

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25 Structure-Activity Relations hip Studies of Peptides Design of Peptidomimetics and Small Molecules Receptor Mutagenesis and Chimeric Receptors The first section reports the synthesis and ev aluation of derivative s of the melanocortin synthetic ligands MTII and SHU9119. It is hypothesized that there may be a cationinteraction between an Arg and the adjacent aromatic ami no acid which may be involved in agonist and antagonist activity. To investigat e this theory, a series of pe ptides was synthesized which incorporate six aromatic amino acids: DPhe, Ph e, DNal(2’), Nal(2’), DNal(1’) and Nal(1’) coupled with either Arg, Lys, or Ala. A cationinteraction is indicate d by the potency series Arg > Lys > Ala. The second section describes the synthesis of a series of 1,4-benzodiazepine-2,5-diones as small molecule ligands of the melanocortin re ceptors. These compounds were designed based on the core tetrapeptide sequence His-Phe-ArgTrp present in all melanocortin agonists. Compounds were synthesized with significan t agonist activity at all receptors. The final section involves the construction a nd characterization of a series of hMC2R (human Melanocortin-2 Receptor) and hMC4R (h uman Melanocortin-4 Receptor) chimeras in order to clarify the unique prope rties of the hMC2R. It is hypo thesized that the MC2R and ACTH may be involved in the homeostasis of human hypertension, and therefore correlated to heart disease and stroke. The study proposed he rein attempts to understand the molecular mechanism of how ACTH stimulates the MC2R. The results of these studies illustrate impor tant intermolecular interactions present in melanocortin ligands which may be used in the future to design more potent and bioavailable drugs.

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26 CHAPTER 1 INTRODUCTION TO DRUG DISCOVERY Rational Drug Design Over the past few decades the focus of drug research has switched from the concept of drug discovery (that is, serendipity ) to that of rational drug desi gn. With advances in molecular biology, genetics, and chemistry, it is now possibl e to design potential lead compounds based on a known biological target. Figure 1-1. Flowchart of drug design from bi ologically active peptid es. Highlighted boxes indicate methods considered in our study. Rational drug design is a met hod for developing potential new therapeutic agents for the treatment of disease based on a known biological target. One common method involves the use of biologically active peptides as a starting point (Figure 1-1).1 This process begins with the identification of a biological receptor system that is stimulated by a specific peptide. Functional assay and competitive binding studies may then be performed to characterize the endogenous ligand-receptor interaction. This is followed by structure-activity relatio nship (SAR) studies in which the molecular basis for the ligand-receptor interaction is probed using substituted peptides

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27 and also by biophysical peptide analysis. This information can then be used to design conformationally constrained pep tides and peptidomimetics to investigate the ideal bioactive conformation required for ligand binding and s timulation. 3-D computer modeling and molecular dynamics studies can also be used to predict liga nd-receptor interactions. These studies can then be used in the design of small molecule ligands with biological activity. An additional receptorbased approach may also be used in which specific amino acid residues in the receptor are mutated to determine which portions of the receptor are interacting with the ligand. This dissertation investigates three experimental methods used to provide information about putative ligand-receptor inte ractions for the design of dr ugs targeting the melanocortin system: Structure-Activity Relations hip Studies of Peptides Design of Peptidomimetics and Small Molecules Receptor Mutagenesis and Chimeric Receptors Structure-Activity Relation ship Studies of Peptides Once a biological target has been identified, it is a common approach in drug design to take the endogenous biological effectors (ie. pe ptides, steroids, small molecules) and submit them to extensive analysis. During this proce ss, the amino acid sequence required for binding to and activating the endogenous receptor is inves tigated and lead peptide sequences optimized, meaning amino acid sequences are m odified in order to produce synt hetic peptides that are more potent than the endogenous peptide. The processes by which peptides are analy zed and sequences optimized are known as structure-relationship studies (S AR) or structure-function studie s. There are many methods to determine the ideal amino acid sequence in cluding the use of truncation studies,2 alanine scans,3

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28 D-amino acid scans,4 enantio scans,5 retro synthesis,5 retro-enantio scans5 and the use of single or multiple amino acid substitution6-9 (Figure 1-2). Figure 1-2. Graphical description of Structure-Activity Relationship (SAR) studies of peptides. A. N-terminal and C-terminal truncation. B. Alanine scan. C. D-amino acid scan. D. Retro scan. E. Enantio scan. F. Retro-enantio scan. Truncation studies. The first method of SAR that is di scussed is truncation or deletion studies (Figure 1-2A).2 This method involves the sequential tr uncation of individual amino acids from either the N-terminus or the C-terminus of the endogenous peptide. As amino acids are removed, functional assay reveals whether or not th e truncated peptide can still elicit a biological response. If there is no change in potency wh en an amino acid is removed, then it may be inferred that that amino acid is not necessary for ligand binding or signaling. If there is a significant decrease in potency upon deletion of an amino acid, then that amino acid may be involved in either ligand bindi ng or signaling. Using this method, the shortest amino acid chain still able to elicit a biological response may be de termined. This is important, as it is desirable for the design of small molecules to know the minimum sequence needed for stimulation.

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29 Alanine scan. Another method common to peptide SAR studies is the alanine scan (Figure 1-2B).3 In this study, each amino acid in the sequen ce is sequentially replace d with an alanine. Alanine is a small, hydrophobic amino acid where R = CH3. When other amino acids are replaced by alanine, the intermolecular interactions between that amino acid side chain and either the receptor or the ligand may be in terrupted. For example, if the pep tide contains a serine that is replaced by an alanine, the hydrogen bonding and hydr ophilicity of the serine are replaced with a hydrophobic group. This, in turn, affects the binding of the ligand to the receptor or the folding of the peptide, causing a change in potency of the ligand. When an amino acid is replaced with alanine and there is a large decrease in potency associated with the substitution, it may be inferred that that amino acid is involved in eith er binding to the recept or or in signaling. D-Amino acid scan. In biological systems, all amino acids are in the L configuration. This means that a chain of amino acids linked by peptid e bonds will often behave in predictable ways when assuming the secondary structure based on the sequence of amino acids.1,10 The peptide will form a series of -helices or -sheets with the side chains of the amino acids projecting in predictable patterns. One method us ed to investigate the effects of a change in configuration on the potency of a ligand is the D-amino acid scan where each amino acid is replaced with the D form of that amino acid, in turn (Figure 1-2C).4 Often, it may be discovered that the ligand is even more potent when specific amino acids are changed to the D configuration. An important example of this phenomenon is seen in the deve lopment of the synthetic melanocortin agonist NDP-MSH from the endogenous peptide -MSH. D-amino acid studies of -MSH revealed that when the phenylalanine residue was replaced with a D-phenylalanine, the resulting peptide was significantly more potent at the melanocortin receptors.4 This change was so significant, that many future synthetic ligands of melanocortin agonists have incorporated the D form of

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30 phenylalanine (Figure 1-3). Incorporation of D-ami no acids is also beneficial in the development of peptides that are more resistant to degrada tion as peptidases in th e body do not recognize the D amino acids as their substrate, resulti ng in a peptide with a longer half-life.4 Figure 1-3. L versus D forms of phenylalanine and effects on -MSH and NDP-MSH11 potency.12 Retro, enantio, and retro-enantio scans. A less used form of studying ligand-receptor interactions through SAR studies is the use of retro, enan tio, and retro-enantio scans.5 A retro scan involves synthesizing the reverse amino aci d sequence of the original peptide (Figure 12D). This switches the N C directionality of the peptide. This reveals information about whether the peptide is only activ e when synthesized in the physio logical direction or whether it is only the sequence of amino acids that matte rs. Additionally, an enan tio study is where all amino acids are replaced with the D fo rm of the amino acid at the same time5 (Figure 1-2E). This completely changes the conforma tion of the peptide. Finally, a retro-enantio scan involves synthesizing the peptide in reverse or der with all amino acids in the D-form5 (Figure1-2F). While not as useful as the truncation studies, alanin e scan, or D-amino acid scan, these methods are often used to explore the effects of cha nges in conformation and peptide direction.

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31 Figure 1-4. Potential ligand-recepto r interactions at the binding poc ket. Blue represents ligand; side chains represen t receptor residues. A. Favorable interaction between aromatic ligand and hydrophobic binding pocket. B. Unfavorable interaction between hydrophobic binding pocket and charged ligand. C. Unfavorable interaction between negatively-charged binding pocket and aromatic ligand. D Favorable interaction between negatively-charged binding pocket and positively-charged ligand. Single or multiple substitutions. Once the important amino acid side chains have been determined by use of other SAR studies, it is possi ble to investigate more specifically the side chain functional groups which are important for ligand-receptor in teractions. In these studies, amino acids possessing different chemical propertie s may be substituted for an important amino acid. For example, each residue may be replaced with a hydrophilic residue (ie. serine), a hydrophobic residue (ie. leucine), an aromatic residue (ie. phenylal anine), a basic residue (ie. lysine), an acidic residue (ie. as partic acid), or with proline, wh ich induces a turn or kink in the peptide chain.10 Information about the intermolecular in teractions between ligand and receptor may be obtained here, enabling the proposal of possible binding pockets in the receptor. For example, if an aromatic or hydrophobic amino acid is well-tolerated and substitution with a charged or hydrophilic amino acid abolishes func tional activity, then it ma y be postulated that this particular amino acid fits into a hydrophobic binding pocket in the receptor (Figure 1-4A,B). Conversely, if charged side chains produce a li gand with good potency, that region of the ligand

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32 may be interacting with a charged or hydrophilic pocket in the receptor (Figure 1-4C,D). These studies are very important in the determination of the ideal peptide sequence needed to bind to and stimulate a target receptor. All the SAR data may be combined and then used to create peptides more potent than the endogenous peptide, or peptidomimetics and/or small molecules which are able to mimic the bioac tive conformation of a given peptide.1 Design of Peptidomimetics and Small Molecules Peptides are very difficult to formulate as drugs because of their high molecular weight, abundance of hydrogen bonding sites, high rate s of degradation in the body and long synthesis.13,14 For these and other reasons, it is often de sirable to develop molecules which mimic the 3-D conformation of important functional gr oups in the active peptide. These molecules which mimic the peptide are often known as peptidomimetics.13,14 Common methods of peptide modification include cyclization, backbone m odification and incorporation of unnatural, constrained amino acids.1 Additionally, turn mimetics may be introduced into the molecule to induce a particular conformation.15-17 Figure 1-5. Peptide torsion angles defini ng secondary structures and conformation. Secondary Structural Features of Peptides Peptidomimetics are often designed to mimi c the secondary structure of a peptide.13,14 There are several second ary structural features common to endogenous peptides and proteins. These are the -helix, -sheet, -turn, and -turn.10,18 These secondary structural motifs are

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33 defined by peptide torsion angles, defined in Fi gure 1-5. The angles are defined as follows: = the angle formed between the NH and the alpha carbon (C ) of the peptide backbone; = the angle formed between C and the carbonyl carbon; = the angle formed between the carbonyl carbon and the NH; and = the angle formed between the first atom of the side chain and the peptide backbone. When the and torsion angles are plotte d against each other, a Ramachandran plot10 is obtained, which depicts the expected secondary structure of the peptide based on the specific and angles. A representative Ramacha ndran plot showing the locations of the -helix and -sheet is shown in Figure 1-610. In this diagram, the white regions correspond to structural conformations that are disallowed to due to steric overlap of atoms, blue regions are allowable in certain situations and red areas correspond to co nformations where there is no steric hindrance. The red re gions correspond to the two most common secondary structural features of peptides and proteins, the -helix and the -sheet. Figure 1-6. Representative Ramachandran plot10 depicting torsion a ngles defining common peptide secondary structural features.

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34 The -helix. The right-handed -helix is characterized by negative phi (-60) and psi (-50) values.10 Hydrogen bonding between the backbone atom s results in a helical structure which repeats every 5.4 .19 There are 3.6 amino acids per turn of the helix and 1.5 rise between each amino acid.19 Each carbonyl oxygen is hydrogen bonded to the NH of the amino acid four residues away (O i to N i+4 )19 (Figure 1-7A). This forms a repeat able, regular, stable structure. Within the helix, all C=O groups point in one di rection, and all H-N groups point in the opposite direction. Side chains extend outward from th e helix and slightly toward the N-terminus.19 Certain amino acids are associated as being compatible with the -helical structure (Table 1-1). These are Ala, Leu, Met, Phe, Glu, Gln, His, Lys, and Arg. Bulky, hydrophobic residues such as Tyr, Trp, Ile, Val, and Thr, and the la rge sulfur atom of Cys do not favor the -helix, and instead prefer the -sheet. Other amino acids such as Gly, Pro, Asp, Asn, and Ser tend to disrupt secondary structures, and are therefore more common in turn structures. The left-handed -helix is much less common due to steric crowding of th e C=O next to the amino acid side chains, and is thus only found in short se gments of Gly-rich proteins. The -sheet. The other most common secondary structur al feature of peptides and proteins is the -sheet.20 In contrast to the -helix, psi values of -sheets are positive, centered at approximately +130.10 Phi values are negative, centered around = -140 degrees.10 Amino acids in a -sheet are spaced 3.5 apart,20 and are arranged in an ex tended, roughly linear manner. The structure is held together by hydrogen bonding between the C=O and N-H of the backbone of adjacent strands.20 These strands can run either parall el or anti-parallel to each other.20 Along a strand, the C=O and N-H are arra nged opposite one anothe r, and alternate side chains point in opposite directions oriented 90o from the backbone20 (Figure 1-7B). Bulky and hydrophobic amino acids such as Tyr and Trp favor the -sheet, as well as branch ed hydrophobics like Ile and

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35 Val, and Cys, which contains a la rge sulfur atom attached to the -carbon of the side chain, making it difficult to arrange in a -helix. Figure 1-7. Common secondary struct ural features of peptides. A. -helix. B. -sheet. Table 1-1. Secondary structural feat ures induced by common amino acids. Amino Acid Secondary StructureComments Ala -helix Cys -sheet Large sulfur group on -carbon Asp Disrupts structure H ydrogen bond on side chain Glu -helix Phe -helix Gly Disrupts structure No side chain to stabilize helix His -helix Ile -sheet Branched at -carbon Lys -helix Leu -helix Met -helix Asn Disrupts structure Pro Turns Kinks chain, causes turns Gln -helix Arg -helix Ser Disrupts structure H ydrogen bond on side chain Thr -sheet Val -sheet Branched at -carbon Trp -sheet Bulky aromatic Tyr -sheet Bulky aromatic

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36 The reverse turn. Many peptides and proteins also cont ain specialized structural features such as turns, which serve to change the direction of the peptide chain.10,18 The most common of these are the -turn and the -turn. The -turn (Figure 1-8A), also known as the reverse turn, serves as a site for chain reversal and may be involved in molecu lar recognition, antibody recognition, receptor binding, or posttranslational modification.18 The reverse turn is defined by the following conditions: positions i i +1, i +2, and i +3 are not in an -helix, and the distance between C ( i ) and C ( i +3) is less than 7.18 There are several types of -turns, the most common being of Type I or Type II. Type I -turns occur when the amino acids in positions i +1 and i +2 are in the L conformation. Type I bond angles are defined as follows: ( i +1)= -60; ( i +1)= -30; ( i +2)= -90; ( i +2)= 0.21 Type II -turns occur when the amino acids in position i +1 is in the L conformation, and the amino acid in position i +2 is in the D conformation. Type II bond angles are defined as follows: ( i +1)= -60; ( i +1)= 120; ( i +2)= -90; ( i +2)= 0.21 The other turn motif is the -turn. This turn is similar to the -turn, except that it only involves three amino acids. A gamma tu rn is defined for three residues i i +1 and i +2 if residues i and i +2 are connected by a hydrogen bond, and the dihedral angles of i +1 fall into one of two categories: classic where = 75 and = -64; or inverse where = -79 and = 69 (Figure 18B).18,22 Like the -turn, the -turn has been shown to be involved in molecular and antibody recognition as well as receptor binding.23

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37 Figure 1-8. Reverse turn structures found in peptides. A. Type I and II -turns along with torsion angles. B. turn and torsion angles. Peptide Modification in the Design of Peptidomimetics Peptide SAR studies are essential in that th ey provide necessary information about the secondary structural character istics and functional groups important for ligand binding and signaling. The next step is to take this informa tion and modify the peptide in such a way as to increase potency and to increase the bioavail ability of the molecule. This is often done by “locking” the molecule into its bioactive c onformation by cyclization or by insertion of constrained amino acids.1 Introduction of unnatural amino acids or mimetics into the peptide chain also serves to make the molecule more resistant to proteolytic degradation in the body by making the peptide less rec ognizable to peptidases.13,14

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38 Global constraints Cyclization. Most endogenous peptides are linear and therefore often very flexible in solution. This gives rise to a la rge number of possibl e conformations that the peptide may adopt. In order to increase the potency of a peptide, it is often desirable to increase the number of molecules which are in the bioactive conforma tion. This can be done by “locking” the peptide into the bioactive conformation in many ways, including cyclization. Endogenous peptides and proteins often contain disulfide bridges between Cys residues to help the protein adopt the correct 3-D conformation. This technique can also be used in solid phase peptide synthesis (SPPS) to bias the pep tide toward the ideal bioactive conformation. The most common cyclization methods are formation of a disulfide bridge or a lactam bridge.1 The disulfide bridge is formed between two cysteine residues contained in the peptide sequence. A peptide may contain one or more disulfide bridge s. This is done through the selective protection and deprotection of cysteine pairs followe d by oxidation (Figure 1-9 and Table 1-2).24 The ease of cyclization depends on ring size and specifi c amino acid composition. Disulfide formation is most often done after cleavage in solution phase, though there are on-resin strategies that may be compatible with certain resins.24 Table 1-2. Methods of disulfide and lactam bridge formation. Cyclization Type Chemistry Solid or Solution Phase? Protecting Group Deprotection Disulfide Fmoc Solution Trt TFA or HF Disulfide Boc or Fmoc Solution Acm Iodine Disulfide Boc Solution Bzl Na/NH3 Disulfide Boc or Fmoc Solution t Bu HF or Hg(II) Disulfide Boc or Fmoc Solution S t Bu RSH or Bu3P Lactam Boc Solid Fmoc Piperidine Lactam Fmoc Solid Alloc/OAllyl Ph3PPd

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39 Figure 1-9. Disulfide Cyclization of Peptides. A. General disulfide formation scheme in which cysteine residues are protected with iden tical groups which may be deprotected separately from peptide-chain protecti ng groups, followed by oxidation to form a disulfide bridge. B. Multiple disulfide formation sc heme used for the synthesis of multiple disulfide bonds on one peptide chain. Each pair of cysteines may be protected and deprotected selectively, en suring correct disulfide pair formation. Another common method of cycliz ation that has gained popular ity in recent years is the lactam bridge.25 This is essentially the formation of a peptide bond between an amine and a carboxylic acid. Lactam bridge cycl izations can be formed in seve ral ways, including head-to-tail (between the Nand C-termini of a single peptid e), head-to-side chain or tail-to-side chain (between a terminal residue or backbone and a side chain), and side chain-to-side chain cyclization (Figure 1-10).25 Current methodology allows for the selective protection and deprotection of side chains to be cyclized on solid phase, and coupling re actions using the same coupling reagents as standard amino acid c oupling (see Chapter 3). Either Boc or Fmoc chemistry may be used by employing suitable protection methods (Table 1-2). Common amino acids used for this cyclization are ones that contain an amine functional group (ie. Lys, Orn, Dpr), or a carboxylic acid (ie. Asp, Glu). Pseudocyclization. Another method to globally constrai n the structure of a peptide is through the stabilization of secondary structural features such as -helices, -sheets, -turns, and

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40 -turns. As was discussed previous ly, these secondary structural features may be important for binding and ligand recognition at the receptor. Pep tides may be designed with a bias toward a particular secondary structure through the insertion of special ized amino acids, or through the incorporation of a small molecule moiety into th e peptide chain which causes the peptide to form a turn structure.15-17 Figure 1-10. Lactam bridge cyclization of peptides. A. Head-to-tail cyclization. B. Side chain-toside chain cyclization. Peptidomimetic and non-peptide libraries have been constructed which incorporate -turns that are melanocortin agonists.26,27 Haskell-Luevano et al reported the synthesis of the first heterocyclic non-peptide molecules that we re melanocortin agoni sts (Figure 1-11A,B).26 These compounds were based on a -turn motif, and were shown to be micromolar agonists at the mMC1R.26 Bondebjerg et al also did a study in which a thioethe r cyclized scaffold was used to mimic the -turn found in the endogenous and synthetic peptides.27 These molecules were shown to be nanomolar agonists at the mM C1R, and the mMC3-5R (Figure 1-11C).27 Recent unpublished studies in the Haskell-Luevano lab have also identified a novel peptidomimetic that contains a -turn and exhibits nanomola r agonist activity at the mM C1R, mMC4R, and mMC5R, but no functional activity at the mMC3R (Figure 1-11F). Recently, Johannesson et al. have identified novel bicyclic motifs (Figure 1-11D,E) that exhibit activity at the Angiotensin receptors.15,16 When an acetal derivative of Asp was incorporated into the Angiotensin II template cyclization occurred toward the C-terminus, and

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41 the compounds exhibited reve rse turn-inducing properties.16 Incorporation of the Glu acetal derivative resulted in cyclization toward the N-terminus.15 Figure 1-11. Small molecules and peptomimetics incorporating and -turn mimetic structures. A,B. First reported melanocortin small molecule agonists.26 C. Thioether cyclized melanocortin agonists.27 D,E. Angiotensin derivatives incorporating a -turn mimetic.15,16 F. Unpublished melanocortin agonist. G. Angiotensin derivative encompassing a -turn mimetic.23 Angiotensin analogues have also been the subject of studies to insert a -turn structure into the endogenous peptides. Figure 1-11G depict s an Angiotensin II analog with an azepine mimetic that has full agonist activity at the AT1 receptor.23 Local constraints Substitution of constrained amino acids. The use of local constraints to bias a peptide towards its preferred bioactive conformation can also be done thr ough the substitution of conformationally constrained amino acids.1 The simplest of these is proline, which tends to introduce a bend or kink in the chain, and removes a degree of rotation from the peptide due to its secondary amine functionality.28 Other cyclic derivatives of am ino acids have been tested as

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42 well and effectively serve to lim it chain flexibility or define cis-trans isomerism.29,30 Figure 1-12 depicts the structures of three conformationally constrained amino acids often used to limit sidechain flexibility. The Tic residue has been shown to selectively adopt the cis isomer, and insertion of this amino acid into -opioid antagonist templates31 and ACE inhibitors32 has resulted in potent and selective compounds. In melanocortin tetrapeptide studies, when Anc was substituted for the His in the Ac-His-Phe-Arg-Trp-NH2 template, the compound was converted from an agonist to an anta gonist at the mMC3R and mMC4R.6 Figure 1-12. Unnatural, constrained ami no acids to control peptide conformation. Backbone modification. Three methods of peptide backbone modification are Nmethylation, use of a CH2NH reduced bond, or substitution with D-amino acids or aza-amino acids.33 Additionally, the N-terminal and C-term inal may be modified by capping groups.34 Both N-methylation (Figure 1-13A) and reduction (F igure 1-13B) serve to reduce the hydrogen bonding potential of the structure as well as make the peptide le ss recognizable to peptidases, and thus more stable. Changing the number of potential hydrogen bonds may serve to change the secondary structure of the peptid e and modify the way it interacts with a receptor. These changes may increase or decrease binding and signaling de pending on the receptor. Nor C-terminal capping with a long aliphatic ch ain (Figure 1-13C) may serve to make the peptide more

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43 lipophilic. This may allow it to cross cell membrane s more easily, or just to disguise it from aminoor carboxypeptidases. A study by Holder et al. showed that capping the N-terminal of the melanocortin His-DPhe-Arg-Trp-NH2 tetrapeptide with an octa noyl group resulted in a compound with nanomolar activity at the mouse melanocortin receptors, and 14-fold increased potency over the endogenous peptide -MSH.34 Finally, Figure 1-13D shows an aza-substituted peptide template, where the general NH2-CH(R1)-COOH amino acid structure is replaced by NH2-NH(R1)-COOH.33 These peptides have a tendancy to form -turn structures, and also help prevent enzymatic degrad ation of the peptide. Figure 1-13. Backbone modifications of peptid es. A. N-methylation. B. Reduction of amide bonds. C. Nor C-terminal capping. D. Aza-substitution. Design of Small Molecules An important aspect of drug design is the de velopment of small molecule templates with biological activity. Although much research is focused on impr oving the oral bioavailability of peptides, it is still difficult to create a peptide dr ug that can withstand the harsh conditions of the digestive tract. Small molecules can be designed th at are more stable to the acid environment of the stomach and able to cross li pid membranes with greater ease. One method that has proved effective in the design of small molecule ligands is based on the principle of “privileged structures.”

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44 Privileged structures. The term “privileged structure” was coined in 1988 to describe selected structural elements that, when functiona lized, have the potential to produce high affinity ligands for more than one type of receptor.35 Extensive analysis of co mmercially available drugs has identified a number of substructures that are able to elicit a biological response at many receptors. A number of these have been depicted in Figure 1-14. The benzodiazepine template has been extens ively studied as a privileged structure.36-40 In fact, this group of compounds was being studied when the idea of privileged structures was first proposed.35 Benzodiazepines have been identified as sedatives, hypnotics, antidepressants, muscle relaxants, antiamnesiacs, and anticonvulsants in the centra l nervous systems, as well as opioid receptor agonists,41 cholecystokinin antagonists,36 oxytocin antagonists,37,38 HIV-Tat antagonists,39 and HIV reverse transcriptase inhibitors40 among many others. This class of compounds is discussed more extensively in Chapter 6. Figure 1-14. Privileged structures of ten used in small molecule design.

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45 Receptor Mutagenesis and Chimeric Receptors The final method of drug design that is discu ssed here is the concep t of a receptor-based approach as opposed to a ligand-based approach. In traditional medicina l chemistry, a ligand is designed to interact with a gi ven wild type receptor system. In this method, a known ligand is tested at mutant receptors to identify important ligand-receptor interactions and to design ligands that may correct for inactivity at naturally-occurring mutant receptors.42 Site-directed recep tor mutagenesis. A common method for generating mutant receptors is site-directed receptor mutagenesis.43 This involves creating point mutations in the receptor DNA sequence that will only change one amino acid residue in the translat ed protein. Often an approach similar to the alanine scan of peptides is used in which key residues are changed to alanine. If binding or stimulation is changed, then that amino acid may be in teracting either with the ligand or in the signal transduction mech anism. Amino acids may also be changed to investigate the presence of speci fic binding pockets in the rece ptor. If there is a hydrophobic binding pocket and one of the side chains is changed to a highly polar or charged residue, then the ligand-receptor interactions may be disturbed and the bindi ng or potency may be altered. Receptor mutagenesis studies of the melanocortin receptors have identified key receptor residues that interact with the endogenous ligands.43-50 These studies have provided valuable information about the nature of the binding pocket in the mMC1R44,45 and the mMC4R,43,46-50 allowing for the design of peptides and small molecules that interact specifically with those areas of the receptor. Chimeric receptors. Another approach to generating mutant receptors involves taking large sections of one receptor and cloning them into another receptor.51 Novel restriction enzyme sites are cloned into each receptor, and then the receptors are digested wi th the given enzymes to form two “pieces” of DNA. The excised DNA can then be ligated into the opposite receptor.

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46 This type of study is valuable because it can be used to identify large re gions of the receptor important for ligand binding and signal trans duction. Sometimes point mutations do not give enough information because it may be the tertiary interactions of large numbers of amino acids working in a concerted fashion to produce the ob served results. Chimeric receptor design can identify regions of each receptor nece ssary for their individual actions. Uses in drug design. The study of mutant receptors is a very important tool in drug design. They can be created to mimic natural polymorphisms found in humans and characterized based on their interactions with natural ligands. Additionally, synthetic ligands can be tested at the mutant receptors to determine if function ma y be restored. A ligand that converts an inactive polymorphic receptor into a functional receptor is a very powerful tool in treating individuals with genetically inactive receptors.

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47 CHAPTER 2 THE MELANOCORTIN SYSTEM The Melanocortin Receptors The melanocortin system (Figure 2-1) consists of five receptors, MC1-5,52-57 which are members of the superfamily of G protein coup led receptors (GPCRs). The MC1R and the MC35R are stimulated by the endogenous agonists -MSH, -MSH, 1-MSH, 2-MSH and ACTH and activate the cAMP sec ond messenger signaling pathway.58 The MC2R is unique in that it is only stimulated by ACTH.53,59 The melanocortin receptors are also antagonized by the only two known endogenous antagonists of GPCRs, Agouti60,61 and Agouti-related protein (AGRP).62,63 Figure 2-1. The Melanocortin system. The MC1R is expressed in melanocytes and is involved in the production of melanin, the pigment responsible for skin and coat coloration.53,64-67 There are two types of pigments, eumelanin and phaeomelanin.68 Eumelanin is the brown/black pigment produced by the binding of -MSH to the MC1R.68 Eumelanin produces a photoprotective effect, reducing damage due to UV radiation.69,70 Phaeomelanin is the red/yellow pi gment produced in the absence of -MSH.68 It has been shown that A gouti is able to displace -MSH at the MC1R, resulting in the production of phaeomelanin.65,71 This makes the indi vidual less likely to tan and heightens the

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48 risk for UV damage.67 It has been shown that -MSH is produced in the skin following UV exposure.72 The MC1R has also been shown to be expressed in monocytes, neutrophils and macrophages, where it aids in the mediat ion of the anti-inflammatory response.73 -MSH antagonizes the effects of proinflammatory cytokines such as IL-1, IL-6, and TNF ,72,74 and downregulates the produ ction of interferon .75 Additionally, -MSH upregulates the production of IL-10, an immunosuppressive cytokine.76 The MC2R is expressed in the adrenal co rtex where it mediates the production of glucocorticoids and aldosterone.53 The MC2R is the smallest melanocortin receptor77 and is unique in that it is only s timulated by ACTH, and not by any of the other melanocortin agonists.53,59 Mutations in the MC2R have been linked to the autosomal recessive disorder, Familial Glucocorticoid Deficiency (FGD).78-83 FGD is characterized by the nonresponsiveness of the body to the actions of ACTH. This results in hypoglycem ia, bacterial infections, and increased skin pigmentation. Pharmacological ch aracterization of the MC 2R has traditionally been difficult due to the fact th at a functional MC2R is only expre ssed in cells of adrenal origin. Cells of non-adrenal origin, such as the HE K293 cell line, expresse s the MC2R on the cell surface, but there is no func tional or binding activity wh en stimulated with ACTH.84,85 Recent studies into this phenomenon have id entified two accessory proteins, MRAP84,86 and Nup 50,87 which have been shown to co-precipitate with the MC2R. MRAP has been shown to restore functional activity to the MC2R receptors when co-transfected into non-adrenal cell lines.86 The MC3R is expressed in the brain, heart, gastrointestinal tract, and placenta,54,88 and has been shown to be involved in energy homeos tasis, though the mechanism of action is not yet known.89,90 Mice who do not express the MC3R have an increased fat mass and a decreased lean mass, although they maintain the same body we ight as their wild type littermates.89,90 In addition

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49 to stimulating the production of cAMP, the MC3R has also been shown to cause an increase in intracellular Ca2+ levels, indicating a poten tial action through induction of inositol triphosphate (IP3) and Protein Kinase C (PKC).91,92 The MC4R is expressed in the brain55,56 and testis, and is involved in energy and weight homeostasis,93 feeding behavior,93 and sexual function.94,95 MC4R knockout (KO) mice exhibit hyperphagia, hyperinsulinaemia, hyperglycemia and increased linear growth when compared to their wild type littermates which leads to severe obesity.93 The endogenous agonist -MSH stimulates the MC4R and results in a decrease in feeding behavior.96 Mice which lack an MC4R are insensitive to -MSH, leading to increased feeding a nd severe obesity. AGRP is able to antagonize the effects of -MSH at the MC4R, resulting in an increase in food intake and body weight.62 Structure-activity relationship studies of -MSH have identified key amino acids involved in ligand binding to the receptor and have aided in the design of synthetic ligands more potent than -MSH at the MC4R. Ligands have also been discovered that exhi bit high selectivity for the MC4R over the other melanocortin recept ors. It is hypothesized that potent agonists selective for the MC4R may be used as therapeutic agents to treat obesity.97 Additionally, the MC4R has been shown to play a role in erectile function in both man and mice.94,95,98,99 Administration of MC4R agonists in mice, such as the synthetic cyclic agonist MTII, results in increased sexual and grooming activity.99 MTII has also been shown to stimulate penile erections in man.100 The MC5R is expressed in peripheral tissues a nd is involved in the secretion of lipids from exocrine glands.57,101 MC5R knockout mice exhibit decreased water repulsion on their skin due to a decrease in sebaceous lipids.101 Additionally, these mice show a reduced capacity to thermoregulate their body temperature.101

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50 The Melanocortin Ligands Agonists. The melanocortin agonists are derived from the prohormone proopiomelanocortin (POMC)102,103 (Figure 2-2). POMC is expresse d in the pituitary gland, skin, immune system, and brain.104-109 POMC is sequentially cleaved at dibasic cleavage sites by prohormone convertases (PC1-2)110,111 into the five melanocortin agonists, -, -, 1-, 2and ACTH, as well as -endorphin.111 In the case of -MSH, C-terminal cleavage by Carboxypeptidase E removes basic ami no acids, followed by N-acetylation by Nacetyltransferase and amidation by PAM (peptidyl -amidating monooxygenase) [reviewed in 112]. The melanocortin agonists can be characterized by the way they interact with each receptor subtype. Table 2-1 shows the potency series for each receptor.113 In all cases, -MSH is the most potent. ACTH is the only peptide able to stimulate all five recep tors, and the only known peptide able to stimulate the MC2R.53,59 Figure 2-2. Posttranslational processing of POMC into the melanocortin agonists. The most well-known me lanocortin agonist is -MSH (Table 2-2). Extensive structureactivity relationship (SAR) studies have been performed on -MSH and have provided valuable information about the intermolecular interacti ons necessary for signaling at the melanocortin

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51 receptors. Truncation studies of -MSH have revealed that the minimal sequence able to stimulate the melanocortin receptors is the tripeptide Phe-Arg-Trp-NH2.114,115 Alanine scans of this peptide have also provided valu able information. Alanine scans of -MSH confirms that the tetrapeptide His-Phe-Arg-Trp is the core se quence of the melanocortin peptides and is responsible for binding to the receptors.3 D-amino acid scans of a peptide can also provide valuable information about the conformation of the amino acid side chains. These studies have shown that replacing the Phe in His-Phe-Arg-Trp with a DPhe results in a dramatic increase in activity at the melanocortin receptors.4 Table 2-1. Ligand potency series fo r the five melanocortin subtypes. Receptor Ligand Potency Series MC1R -MSH = ACTH > -MSH > -MSH MC2R ACTH only MC3R -MSH = ACTH = -MSH = -MSH MC4R -MSH = ACTH > -MSH > -MSH MC5R -MSH > ACTH > -MSH > -MSH Table 2-2. Amino acid sequences of the endogeno us and synthetic melanocortin agonists. The core tetrapeptide sequence Hi s-Phe-Arg-Trp is in bold. These data were used in the design of the synthetic melanocortin agonists, NDP-MSH and MTII (Table 2-2).4,116-118 NDP-MSH is identic al to the endogenous -MSH except the methionine was replaced with a norleucine to ma ke the peptide more stable to oxidation, and Ligand Sequence -MSH Ac-Ser-Tyr-Ser-Met-GluHis-Phe-Arg-Trp -Gly-Lys-Pro-Val-NH2 ACTH(1-24) Ser-Tyr-Ser-Met-GluHis-Phe-Arg-Trp -Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg -Arg-Pro-Val-Lys-Val-Tyr-Pro -MSH Ala-Glu-Lys-Lys-Asp-Glu-Gly-Pro-Tyr-Arg-Met-GluHis-Phe-Arg-Trp -Gly-Ser-Pro-Pro-Lys-Asp 1-MSH Tyr-Val-Met-GlyHis-Phe-Arg-Trp -Asp-Arg-Phe-NH2 2-MSH Tyr-Val-Met-GlyHis-Phe-Arg-Trp -Asp-Arg-Phe-Gly-OH NDP-MSH Ac-Ser-Tyr-Ser-Nle-GluHis-DPhe-Arg-Trp -Gly-Lys-Pro-Val-NH2 MTII Ac-Nle-c[AspHis-DPhe-Arg-Trp -Lys]-NH2

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52 DPhe is used in place of Phe in the core tetrapeptide sequence.4 NDP-MSH has been shown to be more potent at the melanocortin receptors than -MSH.11 NDP-MSH, also known as MT-I, is currently in clinical trials as a therapeutic agent to increase skin pigmentation. MTII is a synthetic cyclic peptid e based on amino acids 4-10 of -MSH.116,117 SAR studies of -MSH were used to identify am ino acids essential for activity.116,117,119 These studies showed that the amino acids in positions 1,2,3,11,12, and 13 were not essential for activity and were removed in the design of MTII. Additionally, Glu was replaced with Asp to shorten the cyclic chain length, Gly was replaced with the basic am ino acid Lys for cyclization purposes, and a Nle was added in place of the endogenous Met4 to prevent oxidation. Previous D-amino acid studies on -MSH revealed that replacing LPhe with DPhe resulted in a much more potent compound, so a DPhe was used in position 7. These studies re sulted in the development of a linear peptide agonist, Ac-[Nle4, Asp5, DPhe7, Lys10] -MSH[4-10]-NH2 that exhibited an increase in biological potency over the endogenous ligand.116 This linear compound was then cyclized through the use of a lactam bridge be tween the side chains of the Asp5 and Lys10 amino acids resulting in the formation of a 23-membered ring. This cyclic peptide, known as Melanotan II or MTII, was shown to be equipotent with -MSH in the frog skin bioassay and 90-100 times more potent than -MSH in the lizard skin assay.117 Prolongation studies show ed that MTII continued to influence biological activity for up to 48 hour s after removal of the compound from the assay media.117 This prolonged response was not observed for either -MSH or the linear Ac-[Nle4, Asp5, DPhe7, Lys10] -MSH[4-10]-NH2 analogs. SAR studies in which the DPhe of MTII was substituted with bulky aromatic amino acids such as DNal(2’) resulted in the generation of a potent antagonist at the MC3R and the MC4R, known as SHU9119.118 SHU9119 is a partial agonist a nd antagonist at the mMC3R, a

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53 competitive antagonist at the mMC4R, and reta ins full agonist activity at the mMC1R and mMC5R.119 ICV administration of SHU9119 to fasted mice and rats causes a dose-dependent increase in food intake which starts slowly and increases over a 12-hour period of time.97 Additionally, co-administration of SHU9119 and MTII results in abolishment of the anorectic effects of MTII, indicating that SHU9119 acts by competitive antagonism of the melanocortin receptors.97,120 MTII administered by intrac erebroventricular (ICV),97,120-122 paraventricular,123,124 or intraperitoneal121,125 routes resulted in a dose-dependent de crease in food intake and a related decrease in body weight in mice.122 It is postulated that this primary effect on feeding is mediated through the MC4R. It has been observe d that when MTII was administered to MC4Rknockout mice, the corresponding decrease in fe eding behavior and weight loss was not observed,125,126 indicating that the MC4R is involved in the mechanism of action. MTII also has been shown to exhibit erectogeni c and tanning effects in humans.100,127,128 MTII has been studied in Phase I and II Clinical Trials in healthy men100 and in men with psychogenic127 and organic128 causes of erectile dysfunction. Figure 2-3. ACTH truncated to -MSH. -MSH represents the first thirteen amino acids of ACTH. Adrenocorticotropin hormone (ACTH) (Table 2-2) is a 39-amino aci d peptide released from the adrenal glands where it is responsib le for the regulation of glucocortocoid and aldosterone release.53,129 Changes in circulating levels of ACTH have been connected with

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54 diseases such as Cushings and Addison’s, as well as Familial Glucocorticoid Deficiency.78-83 ACTH is the only melanocortin agonist which binds to all five receptor subtypes.59 It is the only peptide able to bind a nd stimulate the MC2R.53,59 -MSH represents the first thirteen amino acids of ACTH66 (Figure 2-3). ACTH retains the C-termin al amino acids as well as a basic KKRR tetrapeptide which has been shown to be important for stimulation of the MC2R.130 Truncation studies of ACTH have revealed that the truncated peptide ACTH (1-24) is equipotent with fulllength ACTH.131 Recent studies in our laboratory have test ed truncated derivatives of ACTH at the hMC2R expressed in OS3 cells.130 A cAMP functional assay has shown that deletion of the C-terminal amino acids is tolerated well until the KKRR tetrapeptide sequence begins to be deleted (Table 2-3). Removal of Arg18 results in a 114-fold decreas e in potency and removal of Arg17 results in a 1000-fold decrease in potency; truncation of Lys16 and Lys15 each result in a complete loss of activity at the hMC2R in OS3 cells. These results indicate that there may be unique interactions with ACTH at the hMC2R that are responsible for ligand selectivity. Table 2-3. Truncation study of ACTH tested at the mMC2R.130 mMC2R Peptide Structure EC50 (nM) Fold Difference ACTH(1-24) SYSMEHFRW GKPVGKKRRPVKVYP-OH 1.451.02 1 ACTH(1-23) SYSMEHFRW GKPVGKKRRPVKVY-OH 8.86.0 ACTH(1-22) SYSMEHFRW GKPVGKKRRPVKV-OH 1.190.52 ACTH(1-21) SYSMEHFRW GKPVGKKRRPVK-OH 0.910.38 ACTH(1-20) SYSMEHFRW GKPVGKKRRPV-OH 0.280.07 ACTH(1-19) SYSMEHFRWGKPVGKKRRP-OH 2.391.12 ACTH(1-18) SYSMEHFR WGKPVGKKRR-OH 1.510.79 ACTH(1-17) SYSMEHFRWGKPVGKKR-OH 11.30.8 8 ACTH(1-16) SYSMEHFRWGKPVGKK-OH 16550 114 ACTH(1-15) SYSMEHFRWGKPVGK-OH 1450400 1000 ACTH(1-14) SYSMEHFRWGKPVG-OH >100M No stim ACTH(1-13) SYSMEHFRWGKPV-OH >100M No stim

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55 Less is known about and -MSH (Table 2-2) than the ot her melanocortin agonists. Both peptides contain the core His-Phe-Arg-Trp tetrap eptide shown to be resp onsible for stimulation of the melanocortin receptors. SAR studies of -MSH have shown that this sequence is important for binding, although replacement of Phe with DP he resulted in a decrease in potency.132 Instead, potency was increased by replacing Trp with DTrp.133 Antagonists. The melanocortin system is unique in that it contains the only two known endogenous antagonists of G protei n coupled receptors, Agouti65,71 and Agouti-Related Protein (AGRP).62,63 Agouti (Figure 2-4) is a competitive antagonist of -MSH at the MC1R, MC3R, and MC4R.65,71 AGRP (Figure 2-4) has been sh own to antagonize the action of -MSH at the MC3R and the MC4R.62,63 Additional studies have shown th at AGRP also acts as an inverse agonist at the MC4R.47,134,135 In this capacity, AGRP may be able to regulate cAMP levels in the absence of an agonist.134 When agouti is overexpressed in mice, competitive displacement of MSH by agouti causes the cell to decrease produc tion of the normal brown/black eumelanin pigment, and increase production of red/yellow phaeomelanin,60 these mice additionally show an obese phenotype. Figure 2-4. Partial sequences of the melanocor tin antagonists hAGRP and hAgouti depicting the five disulfide bonds in each ligand. The melanocortin antagonists can be classified as orexigenic peptides, which indicate an increase in food intake upon administration.62,136 Transgenic mice were created which expressed

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56 the human AGRP gene. These mice exhibited increased weight gain compared to their wild type littermates at four weeks of age, as well as an increase in body length and food consumption.62,136 It has been reported that AGRP transgenic mice become obese, exhibiting a phenotype similar to that observed in the MC4R-deficient mice.93 Therefore, it has been proposed that AGRP mediates its effects through the MC 4R in the central nervous system.62 Both agouti and AGRP contain ten cysteine residues which are able to form five disulfide bonds (Figure 2-4).62,71,137,138 One of the most significant core l oops in AGRP is an octapeptide, [Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys].139,140 Of these eight amino acids, three residues have been shown to be critical for ligand binding: these are the Arg-Phe-Phe111-113 triplet,140 which is also found in the agouti protein. As it appears that th e antagonists act by competitively binding to the receptor at the agonist binding site, it has been po stulated that the Arg-Phe-Phe antagonist motif mimics the His-Phe-Arg-Trp motif found in all endogenous melanocortin agonists.140,141 This theory was tested through the design of several peptides. It was shown that a peptide based on the octapeptide loop of AGRP, Tyr-c[Cys-Arg -Phe-Phe-Asn-Ala-Phe-Cys]Tyr [hAGRP (109118)], is a M agonist at the MC1R and an antagonist at the MC4R.141 As the minimal active sequence to show agonist activ ity at the MC1R is the tetr apeptide Ac-Phe-Arg-Trp-NH2,114,115 it has been proposed that the conserved antagonist Arg-Phe-Phe triplet is mimicking the His-PheArg-Trp tetrapeptide found in the melanocortin agonists.141 Additional studies have been performed using chimeric ligands which subst itute the Arg-Phe-Phe triplet of hAGRP(109-118) with the His-Phe-Arg-Trp tetrapeptides of the agonists. When these compounds were tested at the melanocortin receptors, it was shown that th ese compounds are full agonists at the MC1R and MC3R-MC5R, providing further evidence that Arg-Phe-Phe is responsible for antagonist activity.142

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57 Diseases Associated with the Melanocortin System Obesity. Obesity is a serious condition which can cause complications such as type II diabetes mellitus, cardiovascular disease, and hypert ension, as well as increase the risk of certain types of cancer. A recent survey by the Nationa l Health and Nutrition Examination Survey (NHANES) has estimated that the prevalence of overweight and obese individuals in the United States has been steadily increasing since 1960.143,144 The Body Mass Index (BMI) also known as Quetelet’s Index, is the standard measurement for the determination of overweight and obesity and is calculated from the individual’s weight in pounds divided by their height in inches squared multiplied by 703. Quetelet’s Index was used to determine the average ratio of weight and height. A value of 18.5 or less was cons idered underweight, 18.5-24.9 was considered normal, an index of 25-29.9 was overweight, 30-39.9 was considered obese, and an index above 40 was considered severely obese. These values ar e still used today to de termine an individual’s weight status.145 Four national surveys have been conducte d by NHANES since 1960, in which a subset of the population was analyzed according to their BMI.143,144 It was found that since 1994, the prevalence of overweight indi viduals (BMI 25-29.9) has in creased from 55.9% to 64.3%.143,144 The prevalence of obesity (BMI 30-39.9) was 13.4% in 1960, rose to 15.0% by 1980, and in 2000 the prevalence of obesity was 30.9%.143,144 Additionally, the preval ence of extreme obesity (BMI >40) has increased from 2.9% in 1994 to 4.7% in 2000.143,144 As BMI increases, the chance increases for the individual to develop severe health complications. Recent developments in molecular biology, genetics, and chemistry have enabled researchers to identify numerous pathways in the body that contribute to the regulation of feeding behavior and weight homeostasis. It has become evident that the melanocortin system is involved in this complicated network and that th e MC4R is involved in this process. Targeted

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58 disruption of the MC4R in mice leads to hyperpha gia and adult-onset obes ity, suggesting a role of the melanocortin system in the control of appetite and body weight.93 It has been shown that AGRP, an endogenous antagonist of the MC4R62 is able to competitively displace the agonist MSH at the MC4R, resulting in an increase in food in take and weight gain.62 An MC4R-selective agonist may have the effect of d ecreasing appetite and weight gain.97 ACTH-related diseases. The Hypothalamic-Pituitary-Adren al axis is the system through which the brain sends endocrine signals thr ough the bloodstream and to the adrenal glands.146 The signal begins in the hypothalamus, which is a region of the brain responsible for beginning the endocrine cascade by releasi ng hypothalamic hormones. In this case, the paraventricular nucleus of the hypothalamus releases cortic otropin releasing hormone (CRH) into the hypothalamic-hypophyseal-portal system, where it is tr ansported to the anterior pituitary gland. Here, CRH binds to corticotroph cells which re lease ACTH into the bloodstream. ACTH then travels to the adrenal gland, wh ere it binds to melanocortin-2 receptors on the adrenal cortex.53 This stimulates a signal cascade which results in the biosynthesis and rele ase of glucocorticoids such as cortisol.146 Cortisol then acts on the hypothalamu s in a negative feedback system to decrease the production of CRH.146 In this way, cortisol levels are regulated to maintain proper homeostasis. Cortisol is a glucocorticoid released from th e adrenal cortex. In healthy individuals it serves to maintain blood pressure and cardiovascu lar function, slow the inflammatory response, balance the effects of insulin in breaking down glucose, regulate metabolism of proteins, carbohydrates, and fats, help maintain proper arousa l and a sense of well-being, and to help the body respond to stress.146 It is known as the “stress hormone” as it is released during times of acute stress in the “fight or fli ght” response. In this capacity it provides short-term benefits such

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59 as a quick burst of energy, increased memory functions, increased immune functions, and a decrease in pain sensitivity. Ho wever, in times of chronic stre ss, excess cortisol can result in impaired cognitive and thyroid functions, hyperglycemia, and a decrease in bone density, muscle tissue, and immune response, as well as high blood pressure a nd increased abdominal fat which may increase the chances of a heart attack or stroke.146 These symptoms are becoming more common in today’s population due to the high-stress lifestyles many people lead. In these cases increased cortisol is the result of an environmental factor, but there are diseases whic h result in high circulating leve ls of cortisol from biological sources. One example of these conditions is Cushing’s Syndrome. Cushing’s Syndrome, also known as hypercortisolism, is an endocrine disorder caused by chronic exposure to high levels of cortisol.146 It primarily affects indivi duals between the ages of 20-50, and is seen in 10-15 individuals out of every million people per year. Signs and symptoms of this disease include upper body obesity, rounde d face, increased neck fat, thinning of the extremities, thin skin which bruises easily and heals slowly, presence of stretch marks, weak bones, muscle weakness and severe fatigue, as we ll as high blood pressure, high blood sugar, and mental conditions including irrita bility, anxiety, and depression.146 There are many causes for Cushing’s, including pituitary adenoma, which is a benign tumor of the pituitary which secretes excess ACTH, resulting in high cortisol leve ls. It can also be caused by Ectopic ACTH Syndrome, in which tumors outside of the pituitary release large amounts of ACTH, or by adrenal tumors, such as adrenal carcinomas, which secrete excess cortisol into the blood. Familial Cushing’s Syndrome occurs in individuals who have an increased genetic tendency to develop endocrine tumors.146

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60 Some diseases are caused by not enough cort isol or ACTH. Addison’s Disease, or “hypocortisolism” occurs when the adrenal glands do not produce enough cortisol and aldosterone.146 In this condition, individuals suffer we ight loss, muscle weakness, fatigue, hypoglycemia, and low blood pressure. Over 70% of Addison’s cases are caused by autoimmune disorders in which the body destroys the adrenal cortex, reducing cortisol levels.146 The most common cause of Addison’s is a secondary ad renal insufficiency condition caused by the removal of a pituitary or other ACTH-producing tumor, resulting in a lack of ACTH, and thus a lack of cortisol.146 Mutations in the MC2R78-83 have been connected to Familial Glucocorticoid Deficiency (FGD), a rare autosomal recessive disorder. The disease is characterized by nonresponsiveness to ACTH, lowered glucocorticoid production, and elevated serum levels of ACTH.147 The disease normally presents in childhood with the onset of severe hypoglycemia and episodes of bacterial infection. These signs are accompanied by excessi ve skin pigmentation. An understanding of the hMC2R may help in the development of a therapeutic treatment for this disease. Due to the lack of understanding of the mechanism by which ACTH interacts with the hMC2R, it may be difficult to understand the diseas es caused when there is an imbalance in the body. Knowledge of this system may help to tr eat and/or prevent many diseases related to cortisol, leading to a decrease in cardiovascular disease and stroke as well as other syndromes. This may lead to an increase in the quality of life for individuals suffering from these conditions.

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61 CHAPTER 3 GENERAL METHODOLOGIES 1: CHEMISTRY Solid Phase Peptide Synthesis Merrifield Approach Prior to the introduction of the notion of So lid Phase Peptide Synthesis (SPPS) in 1963 by Bruce Merrifield,148 peptide synthesis was accomplished in solution phase.149 Each step required extensive separation, purificati on and characterization, making this method highly laborand reagent-intensive, and fraught with problems such as very low yield and high occurrence of side products. Merrifield worked to solve this problem by creating an insoluble solid support to which the growing peptide is attached during synthesis. The peptide rema ins attached to the resin until cleaved. The method is shown in Fi gure 3-1: Synthesis begins with the attachment of an amino acid which has been protected at the -amine with a temporary protecting group and side-chain protected with a permanent protecting group wh ich is not removed until cleavage. The next amino-protected amino acid is attached to the growing peptide chain after the temporary amine protecting group on the growing chain is remove d. The cycle of deprotection and coupling is repeated until the peptide is the desired length, and then the peptide is cleaved from the resin and the side chain protecting groups removed in one step. This new method of synthesis was far superior to the solution phase reaction for several reasons. First, unused reagents could be simply washed away after th e reaction was complete, eliminating the need for extraction during each step and washing away any non-resin-bound side reaction products. This method also decreased u ndesirable side reactions and provided a way to drive the reaction to completion through the addi tion of excess reagents. Pure peptide yields were also significantly increased over the solu tion phase method as ther e was little loss between steps.

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62 Figure 3-1. General method of solid phase peptid e synthesis including deprotection, coupling and cleavage steps. Successive publications by Merrifield150-152 continued to improve on what is now called the Merrifield approach to SPPS. The Merrifie ld approach (Figure 3-2) makes use of a tert butoxycarbonyl (Boc) protecting group at the -amino group which can be cleaved using trifluoroacetic acid (TFA). Figure 3-3 depict s a possible acid-catalyzed mechanism for the removal of the Boc group. Side chain protecting groups consisted of benzyl-based, acid-labile groups that were stable to TFA, but cleaved in the presence of anhydrous hydrogen fluoride (HF). Coupling was accomplished by the us e of dicyclohexylcarbodiimide (DCC) in dicloromethane (DCM) to form a peptide bond betw een the free amine attached to the resin and the activated carboxylic aci d of a new amino acid.

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63 Figure 3-2. General Boc chemistr y synthesis strategy. Temporary -amino protecting groups are removed with TFA. Cleavage of side ch ain protecting groups and from resin is accomplished using HF. The Boc approach to peptide synthesis was truly revolutionary, a llowing the rapid, clean synthesis of peptides in days which before would have taken months by solution phase approaches. This method has been used ever si nce with great success, although it did not take long for peptide chemists to discover the need for a synthetic method which could be accomplished with milder cleavage conditions, due to the inherent danger and expensive equipment required for HF cleavage. Additi onally, as both the temporary and permanent protecting groups were acid-labile, there was a certain amount of loss experienced due to premature side chain deprotection and cleavage from resin. It was therefore desirable to develop a method that uses different chemistries for the removal of each type of protecting group. Figure 3-3. Proposed mechanism for acid-catalyzed Boc deprotection.

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64 The Fmoc Synthetic Strategy The new method of SPPS was introduced in 1970 by Carpini.153,154 Instead of the graduated acidolysis used in the Merrifield strategy, this method utilized an orthogonal protecting group strategy, where the -amino group was protected by the base-labile 9fluorenylmethoxycarbonyl (Fmoc) group (Figure 3-4). This group could be removed using 20% piperidine in DMF, and the aci d-labile side chain protecting groups were not affected. This allowed selective amino deprotec tion without worry of cleaving th e peptide or its side chain protecting groups prematurely. A dditionally, since no acid was need ed to deprotect, the side chain protecting groups and the li nker between the peptide and re sin could be removed with the milder acid TFA instead of HF. This opened up a wide possibility of options for side chain protection as well as linker functionalization, allowing for the design of linkers that will functionalize the C-terminus of the peptide when cleaved. Figure 3-5 depicts a possible mechanism for the base-catalyzed deprotection of the Fmoc group. Figure 3-4. General Fmoc s ynthetic strategy. Temporary -amino protecting groups are removed with piperidine. Removal of side chain pr otecting groups and cleav age from resin are accomplished using TFA. The new Fmoc method was faster and cleaner than the Boc method and made use of much milder and safer reagents, making it a very popular method for peptide synthesis. Additionally, amino acids were easily functionalized with the Fmoc moiety, which resulted in functionalized

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65 amino acids being available commercially. This also contributed to the popularity of the new SPPS method. Figure 3-5. Proposed mechanism for base-catalyzed Fmoc removal. Resins for SPPS Resins for SPPS are most commonly made of 1% divinylbenzene cross-linked polystyrene.149 This polymer is useful as it readil y swells in common solvents for peptide synthesis such as DMF (N,N-dimethylformamid e), DCM (1,2-dichloromethane), and NMP (Nmethyl pyrollidinone).149 Additionally, it is easily functiona lized with many different types of linkers.149 These linkers serve to act as a tie between the peptide and the polymer support. They can be made to be cleavable by either HF or TF A and different linkers wi ll result in various Cterminal functionalities upon cleavage, the most common post-cleavage functional groups being either an amide or an acid. Table 3-1 shows so me common Boc and Fmoc resins used in this dissertation as well as their cleavage conditions, residual func tionalities, and the type of chemistry it is compatible with. The first amino acid to be attached to the re sin requires different c oupling conditions based on the type of resin used. Th e three common types of resin are hydroxymethyl-based resins, aminomethyl-based resins, a nd trityl chloride resins.149 Attachment of the first amino acid is a critical step, as the initial loading determines the final peptide yield. In complete attachment of the first amino acid lowers yields, and can result in C-terminally truncated peptides.

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66 Table 3-1. Common types of resin used in SPPS. Resin Linker Cleavage Functionality Type of Chemistry Rink-Amide MBHA HF Amide Boc Wang TFA Acid Fmoc 2-chlorotrityl TFA Acid Fmoc Rink Acid TFA Acid Fmoc Rink Amide TFA Amide Fmoc Hydroxymethyl-based resin. For these types of resins, fi rst amino acid attachment is accomplished by esterification of the amino acid to a linked hydroxyl group.149 Examples of this type of resin are Wang resin155 and Rink Acid156 (Table 3-1). The esteri fication reaction is more difficult than formation of an amide bond, as it requires harsher conditions that can lead to problems such as low substitution, enantiomerization,157 and dipeptide formation. The presence

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67 of water increases the possibility of these side reactions, so the reaction should be carried out in anhydrous conditions. Generally DCC or DMAP is used as the catalyst, though these can cause partial racemization of the amino acid, so should only be used in catalytic amounts. It is very difficult to attach the amino acids Cys, His, Pro, Me t, and Trp to these resins, and significant side reactions can occur, so it is recommended that a chlorotrityl-based resin is used if these amino acids are the first to be attached to the resin.158 With the advent of pre-loaded resins, it is possible to purchase the resin with the desired first ami no acid already attached, eliminating the need for the esterification reaction. Figure 3-6. Diketopiperizine formation duri ng synthesis using a hydroxymethyl linker and proline as the first amino acid. When the first amino acid is proline, there is a higher occurrence of premature cleavage by diketopiperazine formation149 (Figure 3-6) due to the unique secondary structure produced by the secondary amine. This results in cleavage of a dipeptide at the beginning of the sequence, and subsequent loss of peptide yield. To prevent this, when proline or N-alkylated amino are used as the first amino acid to be attached, the more steri cally hindered chlorotrityl resin should be used instead.158 Aminomethyl-based resins. Aminomethyl-based resins, such as Rink Amide156,159 (Fmoc) or Rink Amide MBHA (Boc) (Table 3-1) are the eas iest to load with the initial amino acid, as they contain a free amine which may be coupled to the first amino acid using general peptide bond formation. Methods for coupling are discussed later in this section.

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68 Chlorotrityl resins. If the resin to be used is a chloromethyl resin,160 such as the 2chlorotrityl resin158,160,161 (Table 3-1), the esterification pr oceeds by refluxing the desired first amino acid in DMF with the resin for several days One problem with this method is that it is usually not possible to replace all chlorine at oms with the amino acid. If there are any free chlorine atoms, then in the presence of a tertiary amine the Cl can be replaced with the amine, forming a quaternary amine, which will inte rfere with subsequent coupling reactions.158,160,161 It is therefore essential to ensure the resin is fu lly substituted to prevent significant decreases in peptide yield. The advantage of this resin over hydroxymethyl deri vatives is that there is no racemization of the first amino acid observed, and dipeptide formation is eliminated as well.158 This makes them ideal for use when the fi rst amino acid is Cys, Met, Pro, or Trp.158 These resins can also be purchased with the first residue pre-loaded, allowing for standard chain elongation without the first esterification step. It should be pointed out that the 2-chlorotrityl resin is unique in that the linker is hyperacid labile, meaning that very low concentrations of TFA can cleave the peptide in a short period of time. The side chain protecting groups, however, require stronger acidic conditions, meaning that this resin may yield peptides which are still fully side chain protect ed. A fully deprotected peptide may be obtained by longer treatme nt with higher acid concentration. Side chain protecting groups Over half of the common amino acids used in synthesis contain a reactive functionality on the side chain. In order to prevent deleterious side reactions these f unctional groups must be capped during synthesis. The capping groups are normally removed during the cleavage process, but must be stable to reaction conditions enc ountered during the synthesis. For Boc chemistry, the protecting group must be relatively stable to TFA, but must be removed with HF. Fmoc

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69 chemistry allows the use of more acid-labile gro ups which are removed with TFA, but stable to base. Common side chain protecting groups and th e amino acids with which they are most commonly used are presented in Table 3-2. Spec ial protecting groups may also be used which are removed by additional reagents either before or after cleavage. Examples are Acm, which is removed by iodine after cleavage fo r selective disulfide formation,149 and the OAll and Alloc groups, which are removed with a palladium reagent.162 These are generally used for protection of side chains which will be deprotected duri ng synthesis and used to form lactam bridge cyclizations. The lactam bridge can also be formed in Boc chemistry, by protecting with the Fmoc group, which can be selectively rem oved during synthesis using piperidine. Table 3-2. Side chain protecting groups commonly used in SPPS. Type Of Chemistry Protecting Group Abbreviation Cleavage Conditions Amino acids Boc Acm Iodine Cys 3-Bom HF His Bzl HF Asp, Cys, Glu, Ser, Thr, Tyr 2-Cl-Z HF Arg Fmoc piperidine Lys, Asp, Glu For HF Trp Tos HF Arg, His Fmoc Acm Iodine Cys Alloc Pd(Ph3P) Lys, Orn, Dap Boc TFA His, Lys, Orn, Trp OAll Pd(Ph3P) Asp, Glu OtBu TFA Asp, Glu, Ser, Thr, Tyr Pbf TFA Arg Trt TFA Asn, Gln, Cys, His, Ser, Thr, Tyr

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70 Coupling methods The most important part of the peptide synthe tic process is the coupling of one amino acid to another via formation of a peptide bond.149 It is crucial to the success of the synthesis that the coupling method used is reliable, relatively quick proceeds close to 100% completion and is free of deleterious side reactions. In order to ensure good coupling, the new amino acid must be activated at its C-terminus. This involves th e addition of a reagent that will promote the nucleophilic attack of the resinbound amine and release of the activating agent as a leaving group to form the peptide bond. This reagent must al so proceed without loss of chiral specificity of the amino acid (See Figure 3-7 for common enantiomerization reactions of amino acids). Amino acid activation can be accomplished either by the addition of coupling reagents to the amino acid or by purchasing amino acids with an activating group alrea dy attached to the Cterminal of the amino acid. By far the most co mmon method is the addition of coupling reagents which activate the C-terminus.149 These can be divided into two categories, carbodiimide coupling reagents, and onium coupling reagents.149 Figure 3-7. Common side reactions observed duri ng amino acid activation. Carbodiimides. Since Merrifield’s development of SPPS,163 carbodiimides have been widely utilized as coupling reagents along w ith HOBt (3-hydroxybenzotri azole) as the carboxyl activating agent.149 Traditionally, DCC164 (dicyclohexylcarbodiimide) was used, but has been

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71 widely replaced by DIC164-166 (diisopropylcarbodiimide) for it s ease of use and the increased solubility of the urea form ed. Figure 3-8 depicts the possi ble mechanisms of carbodiimidecatalyzed coupling.149 Figure 3-8. Mechanism of amino acid coupli ng using carbodiimide coupling reagents. The synthesis can proceed through two intermed iates, the symmetrical anhydride or the active ester. The reaction goes through the symmet rical anhydride intermed iate when the amino acid: DIC ratio is 2:1 an d there is no HOBt added.149 In the presence of DIC, the O-acyl urea is formed. This intermediate is highly reactive, a nd may rearrange to form the N-acylisourea, which decreases the amount of amino acid available for coupling (Figure 3-8). Attack by another amino acid results in the symmetrical anhydride. This anhydride is then attack ed by the amino group of the resin-bound amino acid, resulting in the formation of an amide bond (Figure 3-9).149 Symmetrical anhydrides are best formed when DCM is the solv ent, though some Fmoc amino acids require the addition of DMF to fully dissolve.149 Additionally, the presence of base should be avoided as this will prevent the initial pr otonation of the carbod iimide and inhibit the

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72 formation of the O-acylisourea. Af ter amino acid activation, base may be added to increase the rate of carboxylate attack and subsequent anhydride formation.149 Figure 3-9. Mechanism of amino acid coupli ng using carbodiimides through symmetrical anhydride formation. Addition of DMF favors the formation of other intermediates such as the active ester or oxazolone, which may contribute to loss of chirality.149 Active ester formation takes place when an activating reagen t such as HOBt (3-hydroxybenzo triazole) or HOAt (1-hydroxy-7azabenzotriazole) are added (Figure 3-10).149 In the presence of these nucleophiles, the active – OBt ester of the amino acid is formed. The –OBt ester intermediate is a very good leaving group, which makes subsequent amino attack and –OBt release relatively fast (Figure 3-11). The concentration of active ester may be increase d with the addition of one equivalent of hydroxylamine. This serves to concentrate th e ester by reacting with the O-acylisourea, preventing the formation of other intermediates. Addition of base afte r amino acid activation may serve to increase the ra te of the coupling reaction.167 Figure 3-10. Structures of common h ydroxybenzotriazoles used in coupling.

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73 Another reactive intermediate that may be formed is the oxazolone (Figure 3-12),149 this is most commonly formed if the amino acid is an N-carboxamide or by attack of a neighboring carbonyl.149 This intermediate is undesirable as it increases the possibility of amino acid racemization (Figure 3-7).149 Addition of a less reactive nucleophile, such as HOBt or hydroxylamines, may be added to reduce formati on of the oxazolone and thus decrease the chances of racemization. Figure 3-11. Proposed mechanism of amino acid coupling through active ester formation using carbodiimides and HOBt as coupling reagents. Figure 3-12. Proposed mechanism of oxazolone fo rmation leading to amino acid coupling when carbodiimides are used as coupling reagents. Phosphonium and aminium coupling reagents. Another widely used coupling method makes use of onium salts.149 These are compounds based on two basic salts, phosphonium and aminium (Figure 3-13), coupled with a benzot riazole. Table 3-3 contains the names and structures of nine common onium coupling reagents. The mechanism for the onium-assisted coupli ng of two amino acids has not been fully elucidated, although it is believed to proceed thro ugh highly reactive intermediates. In the case of phosphonium reagents, the reactive species ma y be an acyloxyphosphonium ion, which goes on to form either a symmetrical anhydride or ac tive ester, depending on the presence of a hydroxybenzotriazole base.165,167-173 Aminium reagents may proceed through the

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74 acyloxyguanidino intermediate, which reacts rapidly with the HOBt in soluti on to form the active ester.168,174-178 In either case, the presence of base in the reaction mixture is essential for the deprotonation of hydroxybenzotriazole. This reaction must be very fast in order to prevent the reactive onium intermediates from remaining in the solution too long and causing unwanted side reactions. One common side reactio n is the N-terminal guanidation of the peptide chain (Figure 3-14) caused by aminium reagents.179 This caps the N-terminal of the peptide chain, preventing chain elongation. To help prevent this, it is good to pre-activate the amino acid with 0.9 equivalents of aminium reagen t before addition to resin.180 Figure 3-13. Structures of phosphonium and am inium salts used in coupling reactions. Figure 3-14. N-terminal guanida tion of peptide chain when HB TU is the coupling reagent. The first of these salts to gain popularity for use in SPPS was the phosphonium salt BOP (benzotriazol-1-yl-N-oxy-tris(dimet hylamino)phosphonium hexafluorophosphate).181,182 One drawback of the use of this salt is th e generation of a highly toxic side product, hexamethylphosphorotriamide,182,183 to avoid this side product, the BOP derivatives PyBOP (benzotriazol-1-yl-Noxy-tris(pyrrolidino)phosph onium hexafluorophosphate)184 and PyBrOP ([bromotris]-(pyrrolidino) phosponium hexafluorophosphate)185 were developed. The aminium reagents HBTU (N-[1H-benzotriazole-1-yl )(dimethylamino)methylamino)methylene]-N-

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75 methylmethanaminium hexafluorophosphate N-oxide),186,187 HATU (N-[(dimethylamino)-1H1,2,3-triazolo[4,5]pyridine-1ylmethylmethanaminium hexafluorophosphate N-oxide),165 TBTU (N-[1H-benzotriazole-1-yl(dimethylam ino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide), and TATU (N-[(dimethylamino) -1H-1,2,3-triazolo[4,5]pyridine-1ylmethylene]-N-methylmethanaminium tetrafluor oborate N-oxide) do not produce this toxic side product. Table 3-3. Phosphonium and aminium-based coupling reagents. Coupling Reagent Abbreviation Structure Benzotriazol-1-yl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate BOP Benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate PyBOP [Bromotris]-(pyrrolidino)phosponium hexafluorophosphate PyBrOP (7-Azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate AOP (7-Azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate PyAOP N-[1H-Benzotriazole-1yl)(dimethylamino)methylamino)methylene]-Nmethylmethanaminium hexafluorophosphate N-oxide HBTU

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76 N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5]pyridine-1ylmethylmethanaminium hexafluorophosphate N-oxide HATU N-[1H-Benzotriazole-1-yl(dimethylamino)methylene]-Nmethylmethanaminium tetrafluoroborate N-oxide TBTU N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5]pyridine-1ylmethylene]-N-methylmethana minium tetrafluoroborate N-oxide TATU Colorometric monitoring methods There are two widely used on-resin colorimetric methods used to monitor the presence or absence of primary or secondary amines duri ng SPPS. The most common method for detecting primary amines is the Kaiser188 test, which makes use of the reag ent ninhydrin, which reacts with free primary amines to produce the purple dye Ruhemann’s purple (Figure 3-15). After Boc or Fmoc deprotection a test is done to detect the presence of fr ee amine (dark blue), and after coupling another test is performed to monitor the completion of the reaction (absence of free amine, yellow color). This method is very usef ul for short (~2-30) amino acid sequences, though its utility decreases for long or aggregated se quences. Additionally, the test does not work for secondary amines such as proline, and the blue co lor is not always seen for amino acids such as serine, asparagine, and aspartic acid.149 The Kaiser test is not suitable for the detection of secondary amines (ie proline) due to the fact that ninhydrin does not interact with second ary amines. To monitor reactions of proline and other secondary amines, the chloranil test189 is used. Chloranil (2,3,5,6-tetrachloro-1,4benzoquinone) reacts with both pr imary and secondary amines to form 2,3,5-trichloro-6-(2-

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77 pyrrolidinyl-vinyl)-[1,4]benzoquino ne (Figure 3-16), which causes the resin beads to turn a green-blue color in acetone. Figure 3-15. Possible mechanism of prim ary amine detection using ninhydrin. Figure 3-16. Complex formed from the reacti on of chloranil with a secondary amine. Solid Phase 1,4-Benzodiazepine-2,5-dione Synthesis The 1,4-benzodiazepines are an import ant class of privileged templates,35 and numerous derivatives have been identified th at have selective activities agai nst a diverse array of biological targets.36-40 Three common types of benzodiazepines are the 1,4-benzodiazepin-2-one (Figure 317A), 1,5-benzodiazepine-2-one (Figure 3-17B), and 1,4-benzodiazepine-2,5-dione (Figure 317C). 1,4-benzodiazepin-2,5-diones have been re ported to possess anticonvulsant, anxiolytic, and antitumor properties, as well as being chol ecystokinin receptor (CCK ), opiate receptor and platelet glycoprotein IIb-IIIa antagonists,190-193 as well as having herbicidal properties.194 In addition, the 1,4-benzodiazepine-2,5-dione core a ppears in a number of natural products, including asperlicin.195-197

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78 Figure 3-17. Structures of co mmon benzodiazepine templates. A. 1,4-benzodiazepine-2-one. B. 1,5-benzodiazepine-2-one. C. 1,4-benzodiazepine-2,5-dione. Solid Phase Organic Synthesis The syntheses of a vast number of organic heterocycles have been described for solution phase synthetic approaches for many d ecades, benzodiazepine synthesis included.192 The application of solid phase synthesis to organi c molecules has only recently begun to gain in popularity.193,194,198-203 Solid phase is much preferable to solution phase for many reasons. The first is that reagents can be easily washed away by filtration after each step, eliminating the need for extensive extraction, purification, and char acterization between each step. This simple washing also removes non-resin bound side pr oducts that may form. Additionally, excess reagents can be added in order to drive the reaction as close to completion as possible, maximizing yield. Solid phase or ganic synthesis can also be performed using the same equipment as solid phase peptide chemistry, resul ting in a low start-up expense for laboratories already doing SPPS. Because the compounds ar e synthesized on resin, a large number of spatially separate molecules can be constructe d in a short period of time with less labor and reagent cost. These compounds can be individua lly cleaved and analy zed in high-throughput applications to generate large dive rse libraries for biological testing. The benzodiazepine privileged structure provide s an ideal framework for application to combinatorial solid phase approach due to th e conserved core structure, many well-defined points of diversity, commercial availability of re agents, and the possibility of multiple points of attachment to resin.192 As benzodiazepines have already been shown to have potential for the

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79 development of drugs, a simple, rapid, and robus t method of synthesizi ng large numbers of compounds is highly desirable. Several solid phase and solution phase me thodologies for the rapid synthesis of benzodiazepine derivatives have been published r ecently. Each synthesis discussed is unique in the starting materials and specifi c methodologies, though most contain the same basic formula: attachment of the first amino acid-based moie ty, acylation, cyclization, alkylation, and finally cleavage from resin. In 1994, Bunin et al. described the synthesis of a 1,4benzodiazepine-2-one library from 2aminobenzophenones, amino acids, and alkylating agents (Figure 3-18).199 A pin apparatus was used in which the 2-aminobenzophenone was coupled to the solid support and the Fmoc protecting group removed. This was followed by coup ling of an amino acid fluoride to the aryl amine. The acid-catalyzed cycli zation was followed by alkylation, and finally cleavage. In this manner 192 separate compounds were synthesize d and tested at the cholecystokinin A receptor.199 Figure 3-18. Bunin et al .199 synthesis of a 1,4-benzodiazepine-2-one.

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80 Figure 3-19. Boojamra et al .194,200 synthesis of a 1,4-benzodiazepine-2,5-dione. Boojamra et al published a synthesis of 1,4-benzodi azepin-2,5-diones in 1995 that also utilized solid support (Figure 3-19).194,200 Merrifield resin was de rivatized with 4-hydroxy-2,6dimethoxybenzaldehyde to yield th e resin-bound aldehyde which was then coupled to an amino acid ester via reductive amination. This formed the tertiary amine necessary for efficient lactamization. Cyclization was base-catalyzed using a lithium salt of acetanilide, which was basic enough to catalyze lactam-ring formati on, but not basic enough to deprotonate other functionalities. Alkylation provide d the final diversity point, a nd acid-catalyzed cleavage from the resin resulted in the desired compound in good yield and purity, with little to no observed racemization. Rapid one-pot solution phase syntheses of benzodiazepines have also been recently examined.192 Keating et al. reported a solution phase synthesi s utilizing the Ugi four-component condensation, which utilizes the combination of a carboxylic acid, an amine, an aldehyde or ketone, and an isocyanide to yield an -acylamino amide (Figure 3-20).192 This compound then

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81 cyclizes in the presence of acid to form a 1,4benzodiazepine-2,5-dione. This is a useful reaction if a solution phase synthesis is desired, how ever it does require the use of column chromatography for purification, and the final com pound is presented as a racemic mixture as the Ugi reaction results in the gene ration of a new stereocenter. Figure 3-20. Keating et al .192 synthesis of 1,4-benzodiazepine-2,5-diones. Figure 3-21. Bhalay et al .201 synthesis of 1,4-benzodiazepine-2-ones. Another method reported by Bhalay et al. makes use of Wang resin on which to build 1,4benzodiazepin-2-ones from amino alcohols, anthranilic esters, acid chlorides, and amines to produce 120 compounds with three poin ts of diversity (Figure 3-21).201 The reaction makes use of amino alcohols, which are synthesized from the corresponding acid chlo ride and anthranilic

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82 ester, and then added to a resin bound acid chlori de. A primary amine displaces a mesylate (L) to give a secondary aryl amine. Cyclization and clea vage take place concurrently with the addition of sodium methoxide and TH F to give the final compound. In the search for short, rapid methods to apply solid phase benzodiazepine synthesis to high throughput applications, Mayer et al. developed a strategy of synthesizing 1,4benzodiazepin-2,5-diones using Wang resin functi onalized with an amino acid, and then reacting this with either an o-nitrobenz oic acid or Fmoc-protected o-anthranilic acids (Figure 3-22).193 Either way, the amine is produced on the aryl ri ng and then a base-promoted cyclization reaction occurs, which causes the molecule to be released from the resin. This results in an increase in final purity, as only compounds which have cycli zed are released from the solid support. The ability to use either nitrobenzoic or anthranilic acids allows for an increas e in diversity over other synthetic methods. Figure 3-22. Mayer et al .193 synthesis of 1,4-benzodiazepine-2-ones. An interesting synthesis was reported in 1999 by Lee et al. which made use of synthesized -substituted -amino esters as the basis for the seven-membered ring (Figure 3-23).202 They

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83 took aryl fluorides with a nitro group ortho to the fluorine and s ubstituted the fluorine with an substituted -amino ester. The nitro group was reduced to an amine, and the ester hydrolyzed. Cyclization in DIC and HOBt provided the desired 1,5-benzodiazepin-2-one. The resulting molecule was alkylated and th en cleaved from the resin. Figure 3-23. Lee et al .202 synthesis of 1,5-benzodiazepine-2-ones. Figure 3-24. Zhang et al .203 synthesis of 1,4-benzodiazepine-2-ones. The final synthesis discussed here was presented by Zhang et al. in 2002 (Figure 3-24).203 This synthesis of 1,4-benzodiazepin-2,5-diones begi ns with either a Rink amide resin or BAL

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84 resin which is functionalized with an aldehyde. The resin is react ed with a primary amine and 4(bromomethyl)-3-nitrobenzoic ac id and then the aryl bromide is displaced with an -amino ester. The nitro group is then reduced and the este r hydrolyzed, and intramolecular lactamization affords the cyclized structure. The compound is alkylated and then released from the solid support by treatment with TFA. Each of these syntheses has its advantages and disadvantag es and provides the framework for different points of diversity. For this work the synthesis by Boojamra194,200 was chosen as the basis for the synthesis of a library of 1,4-benz odiazepin-2,5-diones. This synthesis was attractive due to the available diversity and ease of synthe sis as well as its applicability to solid phase synthesis techniques already in place. The basi c synthesis remained the same, except BAL resin was used, which already had the aldehyde functionali ty attached. This allo wed for the attachment of the amino ester to be the first step, fo llowed by acylation, lactamization, alkylation, and cleavage. Library design and specific methods are discussed in Chapter 6. Colorimetric Monitoring Colorometric tests were used during the benz odiazepine synthesis to monitor the progress of the reaction. The ninhydrin and chloronil tests have been discussed in the previous section.204,205 These tests were used to monitor the conversion of the secondary amine to the tertiary amine after acylation. At the beginning of the synthesis, the reducti ve amination of the amino acid ester and the aldehyde was monitored by the DNPH test for aldehydes (Figure 3-25).206 In this test, 2,4dinitrophenylhydrazine reacts with the resin-bound aldehyde to produce a red color. The reaction is monitored based on the disappearance of the al dehyde, marked by no color change in the test.

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85 The other test used was the bromophe nol blue test for tertiary amines.207 In the presence of a tertiary amine, bromophenol blue changes the resin beads from yellow to a dark green/blue. This test is valuable for monitoring the form ation of a tertiary amine during acylation. Figure 3-25. Reaction of an aldehyde with DNPH to give a red complex. Experimental Details Synthesis of MTII-based CationPeptides All amino acids and reagents were used w ithout further purificat ion. Boc-Ala-OH, BocAsp(OFm)-OH, Boc-DPhe-OH, Boc-His(3Bom)-OH, Boc-Lys(2-chloro-Z)-OH, BocLys(Fmoc)-OH, Boc-Nle-OH, Boc-Trp(For) -OH, Boc-Phe-OH, Boc-D-2-Nal-OH, BocDNal(1’)-OH, and Boc-Nal(1’)-OH were purchased from Bachem (Torrence, CA). Boc-Nal(2’)OH, N-Hydroxybenzotrizole (HOBt), and Benz otriazole-1-yl-oxy-tris -(dimethylamino)phosphonium hexafluorophosphate (BOP) were pur chased from Peptides International (Louisville, KY). N-Diisopropylethylamine (DIEA), pi peridine, anisole, thioanisole, m-cresol and diisopropylcarbodiimi de (DIC) were purchased from Si gma-Aldrich (St. Louis, MO). Dichloromethane, glacial acetic acid, acetonitrile, trifluoroacte tic acid, and ethyl ether were purchased from Fisher (Fair Lawn, NJ). N,N-Di methylformamide was purchased from Burdick and Jackson (McGaw Park, IL). Peptide Synthesis was accomplished by standard Boc chemistry.208 pMBHA Resin (0.28 meq/g substitution) was used. The resin was placed in a manual reaction vessel (Peptides International), and allowed to swell for one hour in dichloromethane. 10% DIEA in DCM was used to neutralize the resin, and a Kaiser test39 performed to detect the presence of a primary

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86 amine. After a positive test ( purple color), the first Boc ami no acid (3-fold excess) was mixed with HOBt (Anhydrous) in 3-fold excess. The mi xture was added to the resin along with a 3fold excess of DIC, and the solu tion mixed with nitrogen gas fo r two hours. After coupling, the resin was washed with DMF twice for one mi nute each, and DCM seven times for one minute each. A Kaiser test was performed after coupling to ensure the absence of any primary amines, indicating that the reac tion had gone to completion. After a negative Kaiser test (yellow solution), the resin was washed with DCM four times for one minute each. The Boc group on the amino terminus of the attached amino acid was next removed (deprotected) using a mixture of 50% TFA, 48% DCM, and 2% anisole (to prev ent the free Boc group from reacting with the peptide chain) once for two minutes, and once for 20 minutes. After the resin was washed with DCM four times for one minute each, and a pos itive Kaiser test obt ained, the resin was neutralized with 10% DIEA and th e next amino acid added in the sa me way as the previous one. This cycle was repeated for all amino acids in the chain. Cyclization of the peptide was performed in solution prior to adding the final amino acid. The Fmoc groups protecting the side chains of the amino acids Boc-Lys(Fmoc)-OH, and Boc-Asp(OFm)-OH, were removed using a 20% piperidine solution in DMF for 30 minutes. After obtaining a positive Kaiser test, the resin was mixed with a 5-fold excess of BOP Reag ent, HOBt (Anhydrous) (or HBTU), and DIEA in DCM at 37oC for up to two weeks in an Advanced Ch emtech automatic synthesizer (440 MOS, Louisville, KY) until a negative Kaiser test was obtained. After the final amino acid was added, the final amino acid in the chain was deprotected, and the resin dr ied overnight in a desiccator. Cleavage by hydrogen fluoride is very dangerous and measures were taken to ensure the safety of the operator. Resin was placed in a Te flon reaction vessel along w ith a magnetic stir bar and the scavengers m-cresol a nd thioanisole in equimolar am ounts (1L scavenger per 2mg

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87 resin). Scavengers are added to react with the free pr otecting groups in solu tion to prevent them from interacting with the newly deportected side chain groups of the peptide. The vessel was attached to the HF vacuum apparatus and chil led in liquid nitrogen. The water and water pump were turned on and all stopcocks opened to check for leaks in the vacuum. If there were no leaks present, the anhydrous HF was stirred with CoF3 and then transferred from the HF reservoir into a Teflon reaction vessel (approximately 3mL pe r 100g of resin). The reaction vessel was removed from the liquid nitrogen (-78oC) and placed in an ice bath to increase the temperature to 0oC, at which point the HF melted. At this point in time, the reaction was left stirring for one hour. After one hour, a Teflon waste trap was placed in liquid nitrogen a nd the HF transferred from the reaction vessel to the waste trap. When all HF had been removed, the reaction vessel(s) were removed from the apparatus and filled with ethyl ether to remove scavengers and form a white precipitate. Finally, the waste trap was removed from the liquid nitrogen and placed in sodium bicarbonate overnight. After addition of ethyl ether to the peptide after cleavage, the mixture was filtered using a fritted glass funnel into a conical flask. The pr ecipitate was washed a second time with ethyl ether. Glacial acetic acid was used to dissolv e the peptide, and filtered into a round-bottomed flask. Filtering with acetic acid was performed three to four tim es using 20mL each time. After all the peptide was collected, the filtrate was fr ozen using dry ice in acetone, and lyophilized to remove the solvent, resulting in a dry powder. 1,4-Benzodiazepine-2,5-diones Synthesis was performed in a manual reaction vessel (Peptides International, Louisville, KY), mixing was accomplished with nitrogen. All r eagents used were ACS grade or better and used without further purification. BAL resin (B ackbone amide linker resin) was purchased from Advanced ChemTech (Louisville, KY). Dichlo romethane, acetic acid, N-methyl pyrollidinone,

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88 acetonitrile, acetone, and methanol were purch ased from Fisher (Fair Lawn, NJ). N,NDimethylformamide was purchased from Burdic k and Jackson (McGaw Park, IL). Sodium triacetoxyborohydride, 1-[3-(dimethylamino)pr opyl]-1’-ethylcarbodiimide hydrochloride, acetanilide, N-butyllithium (10M in hexanes), be nzyl bromide, 2-(bromomethyl)-napthalene, 2phenylbenzylbromide, dimethylsulfide, 1-i odopropane, 1-iodobutane, tetrahydrofuran, triisopropylsilane and trifluoroace tic acid were purchased from Si gma-Aldrich (St. Louis, MO). 2-Amino-4-chlorobenzoic acid and 2-amino-3methylbenzoic acid were purchased from Acros Organics (Morris Plains, NJ ). Arginine-(Pbf) methyl ester, and lysine-(Boc) methyl ester were purchased from Bachem (Torrence, CA). Phenylalanine methyl ester was purchased from Advanced Chemtech (Louisville, KY). Tryptophan me thyl ester was purchased from Fluka (St. Louis, MO). 2-Aminobenzoic acid was purch ased from Alfa Aesar (Ward Hill, MA). Synthesis was performed on solid phase usi ng the method described in Schemes 6-1 and 6-2. BAL resin (0.3 mmol) was swollen in DCM for one hour and tested for the presence of an aldehyde using the DNPH method.206 The first step of the synt hesis involves the reductive amination of an aldehyde and the methyl ester de rivative of an amino acid. Three-fold excess of NaBH(OAc)3 (10 eq, 3.0 mmol) was dissolved in 1 % AcOH/DMF, generating a white suspension. An amino acid methyl ester in 3-fo ld excess (10 eq, 3.0 mmol) was added to the reaction mixture and stirred for 3-4 h. The resin was filtered and washed with DMF (3 x 20 ml), DCM (3 x 20 ml), MeOH (2 x 20 ml) and DCM (3 x 20 ml). A sample of the resin was removed and tested for the presence of a secondary amin e (chloranil test) and the absence of an aldehyde (DNPH test for aldehydes206). The next step involved the acylation of a s econdary amine with a 2-aminobenzoic acid. The resin was dissolved in N-methyl pyrollidi none (NMP) followed by the addition of excess 1-

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89 [3-(dimethylamino)propyl]-1’-ethylcarbodiimid e hydrochloride (EDC). After the EDC was completely dissolved, the 2-aminobenzoic acid wa s added slowly to minimize side reaction that may occur with an unprotected amine, and the r eaction was mixed with nitrogen overnight. The resin was filtered and washed with NMP (2 x 20 ml), DMF (3 x 20 ml), DCM (3 x 20 ml), MeOH (2 x 20 ml) and DCM (3 x 20 ml). A sample of the resin was removed and tested for the absence of a secondary amine (chloranil test) and the presence of a tert iary amine (bromophenol blue test207). Lactamization to form the seven-membered be nzodiazepine ring required the synthesis of a lithiated acetanilide salt. A round-bottomed flas k was oven-dried and acetanilide (24 eq, 7.2 mmol) dissolved in 10 mL THF was added. The flas k was purged with argon gas and cooled to 78oC in a dry ice/acetone bath. A hexane solu tion of n-BuLi (20 eq, 6.0 mmol) was added dropwise over 10 min followed by rapid stirring for 30min at -78oC (Scheme 6-2). After 30 min, 10 mL DMF was added to homogenize the soluti on and the mixture stirred gently for 15 min. The flask was then allowed to slowly come to room temperature. The lithium salt was transferred to to a round-bottomed flask containing the re sin and purged with argon and the resin stirred gently for 30 h at room temperature. Following cyclization, the pH of the solution wa s tested with pH paper (pH~13-14) and an alkylating agent (40 eq, 12.0 mmol) was added and stirred at room temperature until the pH of the solution was approximately 5 as determined by pH paper. The resin was then transferred back to a reaction vessel and washed with DM F (7 x 20 mL), DCM (7 x 20 mL), MeOH (3 x 20 mL), and DCM (3 x 20 mL). The resin was air-dried and then cleaved with a cleavage cocktail of TFA/Me2S/H2O (90:5:5) for 50 h. The cleaved resin was removed by filtration and the filtrate was evaporated and lyophilized.

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90 Ligand Purification and Analysis Ligands were purified by Reverse PhaseHigh Performance Liquid Chromatography (RPHPLC) using a Shimadzu chromatography system with a photodiode array detector and a semipreparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) and lyophilized. The purified peptides were analyzed using RP-HPLC with an analytical Vydac C18 column (Vydac 218TP104). Molecular mass was determined by Mass Spectrometry on a Voyager-DE Pro (University of Fl orida protein core facility). 1H-NMR was performed on a 400MHz Varian instrument using DMSOd6 as solvent and TMS as reference. 1H-NMR of compound 2 was performed on a 600MHz Bruker Advance Console using DMSOd6 as solvent and TMS as reference. Reporter Gene Assay: -Galactosidase Pharmacological analysis was performed by -galactosidase reporter gene assay.209 HEK293 cells stably expressing the melanocortin rece ptors were transiently transfected with CRE/ galactosidase reporter gene. Forty-eight hours post -transfection the cells were stimulated with 100 L peptide (10-4 10-10 M) or forskolin (10-4 M) control in assay me dium (DMEM containing 0.1mg/mL BSA and 0.1 mM IBMX) for 6 h. To the cell lysate plates, 150 L substrate buffer (60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM -mercaptoethanol, 2 mg/1 mL ONPG) was added to each well and the plates we re incubated at 37C. The sample absorbance, OD405, was measured using a 96 well plate read er (Molecular Devices). Data points were normalized both to the relative protein content and non-receptor dependent forskolin stimulation.

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91 CHAPTER 4 GENERAL METHODOLOGIES 2: MOLECULAR BIOLOGY Molecular Cloning Structure of DNA DNA, or Deoxyribonucleic acid, is the thread that holds the fabric of life together. DNA contains all of the information necessary to bu ild every protein in the organism and how the proteins go together to form cells and tissu es as well as synthesi ze non-protein biological molecules. DNA is made up of units known as nucleotides. Each nucle otide consists of a deoxyribose sugar, an organic base, and a phos phate group. Each monomer is linked together through a sugar-phosphate bond, which makes the backbone of the molecule, with bases extending from the backbone as side chains. There are four bases in DNA, these are adenine, thymine, cytosine, and guanine. Adenine and guanine are purines, which consist of a fused ring system, and thymine and cytosine are pyrimid ines, which are single ring molecules. The DNA molecule is made up of repeating nucleotides in a specific order. The order of the nucleotides determines the sequence of amino acids that makes up the protein that gene codes for. Figure 4-1. Structure of DNA and bases. Adenine and thymine base pair and form two hydrogen bonds. Cytosine and guanine form thr ee hydrogen bonds. The sugar-phosphate backbone is shown in black.

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92 Figure 4-2. Formation of a phosphodiester bond dur ing DNA synthesis. The bases pair and DNA polymerase catalyzes the nucle ophilic attack of the 3’OH on the phosphate of the new base, releasing a diphosphate. DNA is a double-stranded molecule. The two st rands of repeating nuc leosides are held together by hydrogen bonds between bases. The bases pair accordi ng to Chargraff’s rules, which state that adenine a nd thymine always pair together, connected by two hydrogen bonds, and cytosine and guanine always base pair form ing three hydrogen bonds (Figure 4-1). For this reason the two DNA strands are an tiparallel and complementary, one running from the 5’ to 3’ direction, and the other running from the 3’ to the 5’ direction. As the DNA molecule is built, each nucleotide base is added through a phosphodiester bond between the 3’ sugar and the 5’ phosphate of the incoming nucleoside (Figure 42). As the DNA molecule grows, it twists, forming the characteristic double helix shape. The DNA double helix was proposed by James D. Watson and Francis Crick in 1953 based on X-ra y diffraction studies of the DNA molecule by Rosalind Franklin.210 Watson and Crick received the Nobel Prize in 1963 for their work with DNA, though Franklin passed away before th e award was presented. The DNA double helix in

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93 its most common form, the B-form,211 is about 22-26 wide.212 Each nucleotide is approximately 3.3 long.212 This results in a structure that unde rgoes one complete turn every 10.4 base pairs.213 The turn of the helix results in tw o grooves, the major groove, which is 22 wide, and the minor grove, which is 12 wide.214 The major groove serves as the recognition site for many DNA-associated transcription factors.215 The B-form of DNA is a right-handed helix. Other forms of DNA are A-DNA, which is a wider right -handed spiral and may be associated with dehydrated DNA, DNA-RNA hybrids, or enzyme-DNA complexes.216,217 Z-DNA is much less common and forms a left-hand spiral.218 The strength of the DNA double helix is based on its C-G content. As the C-G base pair is held together by three hydrogen bonds as opposed to the two hydrogen bonds of the A-T base pair, it is more difficult to separate C and G. Th e relative amount of C-G base pairs may be used to determine the melting temperature (Tm) of a particular DNA strand. This is especially important during the PCR reaction. The C-G cont ent of the hybridization primers determines how strongly they associate with the template DNA, and the C-G content of the template DNA determines how easily the strands de nature between each elongation step. The DNA molecule contains a permanent copy of the genetic code of an organism. The information is encoded in the base pair se quence, and converted in to protein through the processes of transcriptio n and translation. Additionally, the ge netic code is replicated and passed to each daughter cell during mitotic cell division. These important cellular processes are detailed below. Replication of DNA In order to maintain the flow of genetic in formation from one generation to another there must exist a mechanism to create identical copies of a cell’s ge nome to pass on to daughter cells during mitotic division. Before each round of cell division, the entire genome must be replicated

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94 in a reliable manner. Three manners of DNA rep lication are discussed he re, including bacterial replication, plasmid replicati on in bacterial systems, and eukaryotic DNA replication. All replication methods use a similar array of proteins to create a new copy of DNA. In prokaryotes, replication is initiated and car ried out by a series of proteins known as the pre-replication complex. This consists of a he licase (dnaA) which is responsible for unwinding the two strands of DNA, a primase (dnaG or RN A polymerase) which begins synthesis by adding an RNA primer which the DNA polymerases need to start elongation of the DNA strand, and the DNA holoenzyme. This enzyme complex actually pe rforms replication of the DNA and contains DNA polymerases. In bacteria, ther e are two main polymerases, Po l I and Pol III. Pol III is the main polymerase responsible for chain elongation and takes over af ter the primase adds the RNA primer. Pol III cannot in itiate synthesis de novo, and thus re quires a short RNA primer added by the primase, which recognizes the replication start site and adds a short se quence of RNA. Later, Pol I will use its 3’ 5’ exonuclease activity to remove th e RNA primer and replace it with DNA nucleotides. Figure 4-3. Structure of the rep lication fork. Blue boxes represent RNA primers, red arrows represent newly synthesized DNA.

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95 In eukaryotes, replication is initiated by th e Origin Recognition Complex (ORC) which is analogous to dnaA in prokaryotes. The ORC recruits regulatory pr oteins Cdc6 and Cdt1 which then recruit the MCM complex, or Minichromoso me Maintenance proteins, which is a helicaseprimase complex which unwinds the DNA and adds an RNA primer. The primase complex is made of an RNA polymerase a nd a DNA polymerase known as Pol which adds several DNA bases after the RNA. Replicati on is then taken over by Pol Though the proteins are somewhat different, following initiation, DNA replication is similar in both prokaryotic and eukaryotic cells. Figure 4-4. DNA replication of circ ular and linear DNA. A. Replica tion of circular DNA to form two circular daughter strands B. Two replication bubbles forming during replication of linear DNA. Following initiation of replication, elongation take s place at the replic ation fork (Figure 43). Since DNA synthesis only occurs in the 5’ 3’ direction, each strand of DNA is replicated differently. The leading strand is the one which opens in the 5’ 3’ direction, and synthesis occurs continuously. On the other strand, however the replication fork opens in the opposite direction. This strand is known as the lagging strand because synthesis takes place in short pieces known as Okazaki fragments. As the DNA unw inds, new RNA primers are added and DNA

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96 polymerase adds DNA bases until it reaches the previ ous fragment. At this point, in prokaryotic cells Pol I removes the RNA primer and replaces it with DNA bases. DNA ligase then joins the fragments together resulting in a complete DNA strand. As the DNA is unwound, supercoiling at the replication fork is relieved by DNA gyrase in prokaryotes and Topoiso merases in eukaryotes. In both circular and linear DNA, there may be multiple replication forks on each DNA strand (Figure 4-4). This results in more rapid DNA synthesis. Figure 4-5. The rolling circle method of DNA replicati on in prokaryotes. Another method of rapid DNA replication occurs in bacterial systems that contain plasmid DNA. In order to create large numbers of copies of the plasmid, bacteria use a method known as rolling circle replication (Figure 4-5). In this method, one DNA strand is nicked and begins to unwind. The 3’ end of the nicked strand serves as a primer for replicati on of the unnicked strand by Pol III. Replication then proceeds around the ci rcle, displacing the nicked strand as singlestrand DNA. Multiple copies of DNA may be pr oduced as head-to-tail concatemers. These concatemers are converted to dsDNA through leading strand synthesis by Po l III. Pol I serves to remove the RNA primer and then DNA ligase jo ins the ends of the DNA together to create double-strand circular DNA.

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97 Transcription of DNA Transcription is the process by which the in formation in DNA is encoded into messenger RNA (mRNA) for translation into protein. In prokaryotes this ta kes place in the cytoplasm, and in eukaryotes it occurs in the nucleus and then the mRNA is translocated to the cytoplasm for translation. As eukaryotic transc ription is highly complex, the prokaryotic model is presented here. Transcription in eukar yotes is essentially the same only complicated by many more transcription factors and the presence of hete rochromatin which limits the accessibility of the DNA for transcription. RNA, or ribonucleic acid, differs from DNA in two important ways. First is the use of ribose as the sugar in the backbone as opposed to deoxyribose which is used in DNA. Also, in RNA the base thymine is replaced with uracil (U). These differences are shown in Figure 4-6. The base pairing is the same except that ad enine pairs with uracil instead of thymine. Figure 4-6. Structures of DNA and RNA nucleotides. Black arro ws indicate sites of sugar differences between DNA and RNA. The DNA base thymine is shown in blue, and the RNA base uracil is shown in red. A sequence of DNA which is transcribed into a single mRNA is known as a Transcription Unit (TU). The TU consists of upstream promoter units which recruit the transcription machinery to the proper place, the DNA template, and 5’ an d 3’ untranslated regions (UTR). These UTRs are not translated, but are importa nt for regulation of transcrip tion and translation. Since DNA is

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98 double-stranded, there is a coding strand and a te mplate strand. The coding strand runs from 5’ 3’ and contains the exact codi ng sequence. However, this stra nd is not translated as RNA synthesis only proceeds from 5’ 3’ and transcription would resu lt in an mRNA complementary to the coding sequence. For this reason, the oppos ite strand, known as the template strand, is the one transcribed starting at the 3’ end. This result s in an mRNA transcript exactly the same as the coding sequence. Transcription consists of three stages: ini tiation, elongation, and term ination (Figure 4-7). Transcription is initiated by RNA polymerase (RNAP) binding to the promoter upstream of the transcription start site. The RNAP then “m elts” the DNA duplex, opening up a “transcription bubble”, then adds several RNA nucleotides. Elongation is the pro cess by which RNAP processes down the template, adding RNA base s until it reaches the end of the transcript. Transcription may be terminated by one of two methods. In rho-dependent termination, the rho protein causes the polymerase to fall off the template. In rho-independent termination, the transcript forms a hairpin loop followed by a se ries of U’s which destabilizes the RNAP-DNA complex and terminates transcription. Following transcription, the mRNA may be processed to remove introns or modify bases. Figure 4-7. Model of DNA transcription. The coding strand is shown in bl ack and the template strand in red. RNA polymerase (RNAP) s ynthesizes a new strand of mRNA (blue) using the template strand. Translation into protein Following mRNA synthesis, the mRNA template is used to synthesize a new protein in a process known as translation. Due to the complex ity of eukaryotic translation, the prokaryotic

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99 process is detailed here. The main steps of tran slation remain the same between prokaryotic and eukaryotic protein synthesis. Table 4-1. Diagram of mRNA codons translated to amino acid. The fi rst letter is read first on the left, then the second letter across, and the third letter down on the right. The intersection of the three le tters indicates the amino acid coded for by that codon. First Letter Second Letter Third Letter U C A G Phenylalanine Serine Tyrosine Cysteine U Phenylalanine Serine Tyrosine Cysteine C Leucine Serine Stop Stop A U Leucine Serine Stop Tryptophan G Leucine Proline Histidine Arginine U Leucine Proline Histidine Arginine C Leucine Proline Glutamine Arginine A C Leucine Proline Glutamine Arginine G Isoleucine Threonine Asparagine Serine U Isoleucine Threonine Asparagine Serine C Isoleucine Threonine Lysine Arginine A A Start/Methionine Threoni ne Lysine Arginine G Valine Alanine Aspartate Glycine U Valine Alanine Aspartate Glycine C Valine Alanine Glutamate Glycine A G Valine Alanine Glutamate Glycine G Translation occurs in the cytoplasm of the cell, at the rough endoplasmic reticulum (RER). There are four steps of transl ation: activation, in itiation, elongation, and termination. During activation, a tRNA is charged w ith a corresponding amino acid. Tr ansfer RNA (tRNA) is a noncoding RNA which serves to deliver the new amino acid to the ribosome. There are 20 tRNA molecules, each of which corresponds to the 20 natural amino acids. Each amino acid is coded for by a three-base sequence known as a codon. A ta ble of codons and their corresponding amino acids is shown in Table 4-1. Each tRNA contains an anticodon which is complementary to the codon on the mRNA. The codon and anticodon base pair during translation to ensure the addition of the correct amino acid. There are only 20 am ino acids and 64 codons. This is because the

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100 genetic code is degenerate and unambiguous. This means that though an amino acid can be coded for by several different codons, no codon c odes for more than one amino acid. During activation, a tRNA that contains the proper anticodon is charge d with a specific amino acid by aminoacyl tRNA synthetase. Figure 4-8. Model of DNA translation. The ribosome reads the mRNA in the 5’ 3’ direction. New tRNAs with an anticodon complementary to the codon on the mRNA enter into the A site and the attached amino acid forms a peptide bond with the amino acid attached to the tRNA in the P site. Afte r bond formation, the ribosome shifts to the right and a new tRNA moves into the A site. During the initiation step, a ribosome is re cruited to the mRNA to be translated. A ribosome is an organelle made up of rRNA and protein which cataly zes protein synthesis. It is made up of a large and a small subunit which are assembled separately. First, the small ribosomal subunit is recruited to the mRNA st rand by recognition of the ribosome binding site on the mRNA (Shine-Dalgarno sequence in prokar yotes, Kozak sequence in eukaryotes). This sequence serves to properly position the ribosome at the site of the start codon, AUG. This codes for a special Met that has been formylated and is recognized as the universal start of protein synthesis. The large ribosomal s ubunit then binds to the fMet-tR NA and translation is ready to begin.

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101 The ribosome contains three tRNA binding sites: the A site, the P site, and the E site. The A site is the point of entry for new aminoacy lated tRNAs. The tRNA binds to the codon through base pairing with its anticodon. A peptide bond is th en formed between the amino acids attached to the tRNAs in the A site and the P site. The process known as translocation then causes the ribosome to shift three bases toward the 3’ end of the mRNA, moving the tRNA in the P site into the E site and the tRNA in the A site to the P site An empty tRNA in the E site is released into the cytoplasm. A new charged tRNA then moves in to the A site and elongation continues until a stop codon is reached. This process is shown in Figure 4-8. During termination of protein synthesis, a codon is reached which does not code for an amino acid. There are three stop codons, UAA, UGA, and UAG. No tRNA enters the A site causing release factors to catalyze the release of the newly formed peptide and the dissociation of the translation complex. In vivo peptides are synthesized from the N-terminus to the C-terminus. Following synthesis, they may undergo post-translational m odification of specific amino acids to form an active protein. The protein products of translation form the proteo some, which is responsible for all cellular structural and enzymatic processes. The PCR reaction The polymerase chain reaction (PCR) was first described by Kleppe et al in 1971.219 It was not widely accepted though, until 1983 when Kary Mullis popularized th e technique. Mullis received the Nobel Prize in Chemistry in 1993 for his efforts, though patent wars are still pending. PCR is a process by which fragments of DNA may be amplified exponentially. It may be used in the isolation of genomic DNA for purposes of genotyping or establishing a “DNA fingerprint” for an individual. It is commonly used for the amp lification and qua ntification of

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102 plasmid or linear DNA for research purposes, or for the deliberate insertion of specific mutations. Figure 4-9. The PCR reaction. Template DNA is denatured, primers anneal to the desired sequence, and DNA polymerase synthesizes a new strand of DNA. This process is repeated many times resulting in an exponent ial increase in the number of amplified DNA sequences. The PCR reaction requires several component s. The first is a DNA template to be amplified. DNA primers corresponding to the 5’ a nd 3’ ends of the desired sequence are added. These primers are around 20 base pairs long and match the template with high affinity. The G-C content should correspond to about 60% of the base composition to ensure a strong bond. A DNA polymerase is also needed. Th is polymerase must be able to survive the periods of high temperature required for DNA dena turation, therefore polymerases are used from thermophilic bacteria that grow in geysers and are able to withstand temperatures above 110oC. The most widespread polymerase used is from Thermus aquaticus known as Taq polymerase.220 Taq is stable at high temperatures, though prone to errors as it does not possess 3’ 5’ exonuclease proofreading activity.221 A new polymerase was isolated from the thermophilic archaeon Pyrococcus furiosus ( Pfu ).222 Pfu is superior to Taq as it contains a high fidelity 3’ 5’

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103 proofreading exonuclease system. This results in substantially fewer PCR-induced mutations. The remaining ingredients in the PCR mi xture include dNTPs (deoxyribonucleotide triphosphates) from which to build the new DNA, a suitable buffer for maximal activity of the polymerase, divalent cations such as Mg2+ or Mn2+, and potassium ions. The general PCR reaction consists of six main steps (Figure 4-9). First is an initialization step of 94-96oC to activate the polymerase. This is followed by a denaturation step of 94-98oC for 20-30 seconds to denature the DNA. An annealing step at 50-65oC for 20-40 seconds is then performed to allow the primers to bind to thei r complementary sequences on the template. The annealing step should be performed at only a fe w degrees above the melting temperature of the primers to ensure that only sequences with high fidelity matches bind to the template. Annealing is followed by an elongation step at the optimal temperature for the polymerase, usually around 75-80oC. The length of this step varies with the le ngth of the desired amp lified sequence. It may be assumed that the polymerase will add a bout 1000 bases per minute. The denaturation, annealing, and extension steps are repeated 20 -30 times. Each new DNA sequence may serve as a template for the next cycle resulting in expone ntial increases in the number of amplified DNA sequences. After the last cycle, a final elongation step is performed to ensure the extension of any remaining single-stranded DNA. The P CR mixture may then be held at 4oC for short-term storage. The PCR reaction has become an indispensable t ool in molecular biology and forensics. It has opened the door to a vast realm of po ssibilities in DNA mani pulation and cloning. Restriction endonuclease digestion A Restriction Enzyme, also known as a Rest riction Endonuclease, is an enzyme which recognizes specific DNA sequences and cuts the DNA backbone at specific locations without interfering with the base. The term “restriction” came from the observation that E. coli seemed to

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104 be restricting infection by bacteriophages and it was inferred that this was a mechanism used to resist viral attack. Further resear ch revealed a plethora of bacterial enzymes that could be used to cut DNA at specific sequences. In 1978 Daniel Nath ans won the Nobel Prize for his discovery of restriction enzymes which led to the development of recombinant DNA technology. Restriction enzymes generally recognize sequences of four, five, six, or seven bases. Often these sequences are palindromic, this means that both strands read the same from the 5’ 3’ direction. Restriction fragments ma y have blunt ends or sticky e nds. Blunt ends means that the enzyme cuts at the same place on both strands. These fragments may be ligated to any other blunt end fragment. Sticky ends result from an enzyme which cuts at tw o ends of the recognition sequence, resulting in a single-stranded overhang. These fragments may be ligated with another DNA fragment that has been cut with the same en zyme. DNA ligase is used to repair the breaks in the backbone and create a new DNA sequence. One of the main uses of restriction enzyme s is the cutting of a fragment of DNA and religating into a plasmid, known as “cloning.” The plasmid can then be inserted into bacterial vectors or mammalian cells for expression of the desired gene. In this way restriction enzymes have played a vital role in the identification and manipulation of a vast number of genes and have made the process of DNA recombination and cloning possible. Gel electrophoresis Gel electrophoresis is a method used to separate DNA fragments of differing size and conformation. It is based on the principle that when DNA is placed in neutral or alkaline conditions, the phosphate groups become ionized giving DNA an overall negative charge. When a current is applied to the negatively char ged DNA it will migrate towa rds the positive anode. Two types of gels are commonly used, agarose a nd acrylamide. As acrylamide is toxic, agarose is more commonly used. Agarose is purified from agar, which is a polysaccharide found in the

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105 cell walls of some types of algae and seaweed (F igure 4-10A). Agarose is neutrally charged and forms large pores that make it ideally su ited for separation of large biomolecules. Figure 4-10. Reagents for gel elec trophoresis. A. Structure of the crosslinking unit of agarose. B. Structure of ethidium bromide. A sample of DNA is loaded onto a gel along with a loading buffer. The loading buffer contains a denaturing agent such as SDS or urea to prevent any enzymatic reactions, glycerol to weight down the DNA at the bottom of the well so it does not come out, and dyes which are used as mobility markers. These markers are common ly bromophenol blue and xylene cyanol. They migrate with the DNA for a visual representation of the progress of the DNA down the gel. The gel is run in a buffer such as TAE (Tris-acetate EDTA buffer) which carries the charge from the anode and cathode through the gel. DNA migrates through agarose gels based on its size and conformation. Large pieces of DNA will not travel as fast as small fragments, allowing for size separa tion. Restriction enzyme digests of plasmids or DNA segments can result in characteristic gel patt erns based on the size of the fragments. The conformation of a DNA molecu le also determines its movement through the gel. Circular DNA will move fast er than linear DNA, allowing fo r differentiation between nicked and circular plasmids.

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106 Following DNA migration through the gel it is necessary to view the DNA. Since DNA is invisible to the naked eye, a chemical known as ethidium bromide is commonly used to view the DNA bands. Ethidium bromide (Figure 4-10B) is a carcinogen which intercalates between the DNA bases. When exposed to UV light it fluoresc es orange, allowing for the visualization of DNA. The DNA bands may be cut out of the gel a nd the DNA purified for further experiments. Sequencing An important part of DNA mani pulation is sequencing. It is often useful to know the exact sequence of nucleotides. In the seventies Sanger et al. developed a method for chain-termination sequencing.223 In this method, four tube s are used. Each tube is loaded with template DNA, primers, and regular dNTPs, but each tube also gets a ddNTP (dideoxynucleotide triphosphate) corresponding to one of the four bases. Incorpor ation of the ddNTP results in chain termination as there is no 3’OH to continue building the ch ain. The reaction mixtures can then be run on a gel. Based on how far each band travels it can be inferred how long the chain is. As only one ddNTP is added to each tube, it is known that the last base corresponds to that ddNTP. A more recent method automates this process an d only requires one reaction mixture. It is known as the Big Dye Terminator (BDT) method. Each ddNTP is coupled to a donor and an acceptor dye. The donor dye is 6-carboxyfluorescein (6-FAM). When coupled to different forms of rhodamine, the dyes fluoresce at different wave lengths, allowing for the identification of the terminal base. The acceptor dyes are commonly as follows: dichloro-R6G (dichloro-rhodamine 6G) attached to adenine, dichloro-ROX (dichl oro-rhodamine X) for cytosine, dichloro-R110 (dichloro-rhodamine 110) is attached to guanine, and dichloro-TAMRA (dichlorotetramethylrhodamine) is used for thymine. The BDT reaction mixture, template DNA, a sequencing primer and buffer are mixed and amplif ied using PCR. As the chain grows, the dyelabeled ddNTPs are incorporated resulting in a mixture of DNA se quences of every length. After

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107 PCR, the purified PCR product is run on a special ized machine which runs the reaction mixture through a special polymer. As w ith gel electrophoresis, small DNA fragments run faster than large segments. As each fragment passes through an argon ion laser, the 6-FAM donor dye is excited, which then transfers its energy to the ac ceptor molecule. A detector reads each dye and differentiates between the dyes. A computer prog ram then interprets th e fluorescence and assigns a specific base to each position and plots th e sequence on an electrophe rogram (Figure 4-11). This sequence can then be used for seque nce confirmation and further experiments. Some drawbacks to this method include ineffi cient ddNTP incorporation or “dye blobs” resulting from clumping of the dyes and causi ng artifacts in DNA traces. These problems are overcome through use of highly efficient polymera ses and through continual development of the chemistry used. Though expensive, BDT sequencing is highly reliable and produces high fidelity sequences. Figure 4-11. Model showing how BDT sequencing is read by the detector. Sequences are run through a polymer which separate the sequences by size. As they pass by the detector, the fluorescent label on the 3’ end is read and the base identified. Bacterial Expression Vectors Escherichia coli E. coli is a gram-negative, rod-shap ed bacterium of the family Enterobacteriaceae that is most commonly used in genetic manipulation. It is used because its

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108 genome is well-characterized and it is easy to grow in the laboratory. The bacteria are ideal for use in plasmid amplification and can be ma de chemically competent for transformation. E. coli grows well in LB-media (Luria-Bertani) and can be grown either on agar plates or in broth suspensions. E. coli was discovered in 1885 by the German pediatrician and bacteriologist Theodor Escherich. The bacteria are commonly found as part of the normal flora of the mammalian large intestine, and thus is an importa nt indicator organism for fecal contamination. Some strains can cause food poisoning, su ch as the publicized O157:H7 strain. The strain used in this work is the DH5 strain of E. coli This is a strain that is competent for transformation. It is sensitiv e to antibiotics such as ampici llin and kanamycin so plasmids containing resistance genes may be transformed fo r antibiotic selection of transformed bacteria. Figure 4-12. Bacteria l growth curve. The bacterial growth curve (Figure 4-12) is common to most proka ryotic species. A new culture starts with the lag phase, where there is l ittle growth until the bact eria acclimate to their new environment. This is followed by the exponen tial phase where bacteria l growth occurs very rapidly. When media resources become depleted th e bacteria enter a sta tionary phase where the bacteria are alive but do not di vide. Finally, if no new nutrien ts are added, the population will reach a death phase where the bacteria begin to di e. For plasmid amplifica tion it is necessary to maintain the culture in the expone ntial phase for optimal results.

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109 Cloning plasmids. Plasmids are small, circular, doublestranded DNA that exist outside of the chromatin and are stably inherited. They may be used to insert gene s of interest into both bacterial and mammalian cells. Most synthetic plasmids contain ge nes that confer resistance to certain anitibiotics to allow selection of ce lls based on their incorporation of the plasmid. There are several characteristics of an ideal cloning plasmid. The first is that it should have an easily selectable phenotype. This allows for di stinction between cells that express the plasmid and cells that do not. Plasmids should also have a large number of unique re striction sites. Most plasmids have a multiple cloning site (MCS) that contains a large number of restriction sites not found anywhere else in the plasmid. This allows the researcher to match the restriction enzymes with their gene of interest. Plasmids should also be fairly small to allow for greater stability and ease of transformation. Finally, the plasmid should ha ve a relaxed replicati on control to allow it to replicate in large quantities in bact erial vectors for rapid amplification. Several plasmids are used in this wor k. These are pBK-CMV, pcDNA3, and pMV-CRELuc. pBK-CMV is a bacterial plasmid useful for site-directed mutagenesis. The gene is cut out and replaced into pcDNA3 before transfecti on into mammalian cells. pcMV-CRE-Luc is a reporter gene plasmid transiently tr ansfected before functional assays. Transformation. Transformation is the process by which plasmid DNA is taken up by bacterial cells and replicated for plasmid pur ification. The process is detailed as follows. Competent bacteria are placed on ice and a small quantity of plasmid DNA is added to the cells. The bacteria are then incubated on ice for 2030 minutes while the bacteria take up the DNA. The bacteria are then submitted to heat shock for 45-60 seconds at 42oC. This causes them to produce heat shock proteins so they don’t expel the foreign DNA right away. They are incubated on ice for another 2 minutes to stop the heat sh ock process and then rich SOC media is added

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110 and the bacteria are gently shaken at 210 rpm at 37oC for at least an hour. During this time the bacteria begin to grow. They are then plated on LB-agar plates supplemen ted with the antibiotic of choice and grown overnight at 37oC. If the bacteria have taken up the plasmid DNA they will be resistant to the antibiotic and colonies will form on the ag ar in about 12-16 hours. Single colonies can then be subcloned into an antib iotic LB broth and grown for plasmid isolation. Plasmid purification. In order to obtain enough plasmid for sequencing, restriction digests or transfection, the plasmids ar e amplified by inserti on into a bacterial e xpression system, as described above. For small-scale reactions, a Miniprep kit from Qi agen was used. This provided about 30 L of 0.3 g/L DNA for sequencing or di gestion. For large-scal e isolation reactions, the Maxiprep kit from Qiagen was used to provide anywhere from 100-2000 L of 1-2 g/L DNA which may be used for any application incl uding stable transfection into mammalian cells. The general procedure is the same for both sm alland large-scale reactions. A quantity of bacteria expressing the desired plasmid is grow n in LB broth supplemented with ampicillin overnight at 37oC and 275rpm. Bacteria are collected by centrifugation and growth media is discarded. The bacteria are resuspended (QiagenBuffer P1) and then subj ected to alkaline lysis (QiagenBuffer P2). The lysi s solution contains a NaOH-SDS solution in the presence of RNaseA. SDS, or sodium dodecylsulfate, is used to solubilize the proteins and phospholipids of the cell membrane, leading to membrane breakage and release of the cell contents. NaOH is used to denature the chromosomal DNA, plasmid DNA, and proteins to prevent enzymatic reactions. Following lysis, a neutralization buffer (QiagenBuffer P3) containing acidic potassium acetate is added. The high salt concentra tion precipitates KDS (potassium dodecylsulfate), trapping the cellular debris in a salt-detergent complex. Th is debris contains pr oteins, chromosomal DNA, and other cellular components. The plasmid DNA, however, remains in solution as it is smaller

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111 and covalently circular, allowing it to renature quickly after pr ecipitation of the detergent. The precipitate is removed by centr ifugation and the lysate containi ng plasmid DNA is applied to a column containing a specialized resin (Qiagen) which selectively bi nds plasmid DNA. The column is washed with a medium salt buffer (Qia genWash Buffer QC) which serves to remove contaminants such as degraded RNA and prot eins without affecti ng the bound plasmid DNA. The wash buffer also disrupts non-specific intera ctions of proteins with the DNA allowing for removal of nucleic acid binding proteins withou t having to use phenol. Ethanol in the buffer disrupts any non-specific hydrophobic inte ractions resulting in an in creased purity of the plasmid DNA. At this point, the DNA is e ither eluted with a high salt buffer (QiagenBuffer QF) in the case of a Maxiprep, or water for Minipreps. Th e Miniprep DNA is ready for use; however the Maxiprep undergoes several more purification steps. Isopropanol is added to desalt and concentrate the DNA, and then centrifuged. The DNA is then washed with ethanol, which is easier to remove than isopropanol and then resuspended in a suitable buffer such as TE Light (pH~8.0). DNA concentration may then be determined by spectrophotometric methods or by agarose gel electrophoresis. The DNA is ready to use for any experimental application needed. Cell Culture and Transfection Mammalian Cell Lines The HEK293 cell line. The HEK293 cell line was develope d in the early seventies in the laboratory of Alex Van der Eb in Leiden, Holland.224 A culture of human embryonic kidney cells were transformed with sheared DNA from the adenovirus 5 virus.224 Later it was shown that the transformation was brought about by incorporation of a 4.5kb insert from the left arm of the viral genome into chromosome 19 of the cells.225 This resulted in an immortal cell line which is able to grow in solution without changing for an i ndeterminate length of time. Further studies on the

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112 origin of the HEK293 cell line indicate that it is possible that the line originated from a neuronal cell present in the kidney culture that was transfected.226 HEK293 cells were quickly shown to be a va luable cell line for molecular biology and biotechnology applications. They are easy to grow and transfect with cl ose to 100% transfection efficiency with the calcium phosphate precipitation method.227 Though the cell line is not a good model for normal or cancer cells, it has become extremely useful in protein expression and functional assays. Its high transfec tion rates make it ideal for transf ection of a gene of interest and subsequent protein expre ssion analyses. In the biotechnol ogy industry, HEK293 cells have become valuable as a way to produce therapeu tic viruses and protei ns for gene therapy applications. The OS3 cell line. The OS3 cell line was derived fr om the murine Y1 adrenocortical tumor cell line along with the Y6 cell line. Thes e cell lines contain a wild type MC2R gene, however this DNA sequence is not functional du e to growth in the presence of forskolin.228 The cells do, however, contain an int act cAMP signal transduction pa thway, resulting in a functional transfected MC2R.228,229 This cell line is used as it is ab le to produce a func tional transfected MC2R, however its drawbacks are th at it grows slowly and is very difficult to transfect with a gene of interest (<2% efficiency). Transfection Transfection is the process by which foreign DNA is introduced into a eu karyotic cell. This is often done to test gene expression or functionality and is an important tool in molecular biology. Once inserted, the gene may be transi ently or stably transfected. In transient transfection, the plasmid containing the gene is expressed for only one or two generations, and eventually is lost during mitotic divisions. Th e gene expression is initially very high but transfection levels vary between experiments. The gene may also be stably transfected. In this

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113 type of transfection, a selective pressure is placed on the cell to force it to retain the plasmid and stably express the gene for a long period of time. The antibiotic Geneticin, or G418, is often used for this process. G418 is an ami noglycoside antibiotic isolated from Micromonospora rhodorangea It is toxic to both prokar yotic and eukaryotic cells, bu t resistance may be inferred through transfection of the neo gene, which is of ten present in plasmids designed for mammalian transfection. Once stable expression is achieved, the cells produce the protein of interest in stable quantities for a long period of time. Many methods exist for the transfection of DN A into eukaryotic cell lines. The most common and cost-effective is the calciu m phosphate method developed by Graham et al .227 In this method, a HEPES-buffered saline solution co ntaining phosphate ions is combined with calcium chloride and the DNA to be transfecte d. A precipitate of calcium phosphate forms, binding DNA. Through a mechanism that is not fully understood, the cell takes up the precipitate along with the foreign DNA. Another method involves the use of lipid-base d reagents. These reagents form liposomelike structures around the DNA. The liposomes bi nd to the cell membrane, releasing DNA into the cell. This process is often known as lipofection, and the formul as for the reagents is highly proprietary. Lesser used methods for transfection include electroporation, heat shock, magnetofection, use of the gene gun, in which DNA is coupled to an inert nanoparticle and then “shot” into the nucleus, as well as the use of viral DNA carriers. Functional Assay Pharmacology Definitions A discussion of functional pharmacology re quires an understandi ng of the physical graphical representation of the data. Examples of these graphs are shown in Figure 4-13. Figure

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114 4-13A depicts some agonist curves. The log concen tration of the ligand is shown in the x axis and the percent stimulation in the y axis. Curve A (red) depicts a typical full agonist. Curve B (green) shows a full agonist that is less potent than A. As the pot ency decreases, the curve shifts to the right. Curve C (purple) is an example of a partial agonist. A partial agonist has the same shape as a full agonist and a maximum stimulation is reached, but the maximum is less than 100% when compared to forskolin control. Cu rve D (red) shows a ligand that has no agonist activity, and Curve E (black) is an example of an inverse agon ist. An inverse agonist is a compound that is able to decrease cAMP levels below basal levels. In this work antagonist values ar e obtained using a Schild analysis.230 This involves costimulation of the receptor with a known agonist and a susp ected antagonist. The curve is shown in Figure 4-13B. The blue represents th e agonist, and the green curves represent the agonist combined with increasing concentrations of antagonist. If the compound is an antagonist, then increasing concentrations will result in a shif t to the right of the agonist curve. This assay tests for competitive antagonists only. It is eff ective because a competitive antagonist competes with an agonist for the same binding site. Wh en the antagonist binds however, there is no receptor stimulation. This gives the appearance of a less potent agonist. If no right shift occurs when antagonist concentration is increased, then the compound is not an antagonist. Sometimes a ligand might have what is known as mixed pharmacology. In this case the ligand may have some partial agonist activity but also have anta gonist activity. These compounds are not well understood but may be important for conti nued receptor or ligand characterization. Figure 4-13C shows how an EC50 value is determined. An EC50 value is the concentration of a ligand which results in 50% stimulation of th e receptor as compared to forskolin control. This value is widely used to de scribe the functional activity of a ligand at a particular receptor. A

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115 higher EC50 value suggests a less potent ligand (shi ft to the right) while a smaller EC50 value suggests a more potent ligand (shift to the left). Figure 4-13. Pharmacology curves. A. Shows va rious agonist curves. Curve A = full agonist, Curve B = less potent full agonist, Curve C = partial agonist, Curve D = no agonist, Curve E = inverse agonist. B. Model of a Schild analysis. Blue curve is the known agonist. Green curves represent the agonist costimulated with increasing concentrations of antagonist. C. Determination of EC50 value from a dose response curve. Luciferase Assay Firefly luciferase from the North American firefly Photinus pyralis has been extensively studied for its role in bioluminescence.231 The endogenous substrate of lu ciferase is luciferin, and the reactions catalyzed by luciferase are as follows: Luciferase + Luciferin + ATP + Mg2+ Luciferase + Luciferyl-AMP + PPi Luciferase + Luciferyl-AMP + O2 Luciferase + Oxyluciferin + AMP + CO2 + hv The reaction begins with the formation of enzyme-bound luciferyl-AMP which then is oxidatively decarboxylated to form oxyluciferin and light. The yellow-green light is emitted at pH 7.5 to 8.5 at a peak emission of 560nm. When the luciferase enzyme is supplemented with excess luciferin, ATP, and oxygen, studies have show n that the light produced is proportional to

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116 the amount of luciferase in the solution.232 It is this property of the enzyme that allows for its use in quantitative assays. Prior to the development of the luciferase assay, the CAT (bacterial chloramphenicol acetyltransferase) enzyme was used as a me thod of monitoring eukaryotic promoter activity.233,234 The luciferase assay, however, is superior to this method as it is less expensive, more sensitive, and does not require the use of radioactive substrates. Figure 4-14. Luciferase reporte r gene assay. When a ligand st imulates the GPCR, G protein activates adenylate cyclas e which converts ATP to cAMP. cAMP binds to CREB which binds to CRE units on the reporter gene causing transcription of the luciferase gene. The cell makes luciferase protein. When the luciferase substrate D-luciferin is added, light is released. The luminescence can be read on a luminometer. Here, dark yellow indicates a high level of luminescence and light yellow low levels. The luciferase reporter gene assay is descri bed in Figure 4-14. A plasmid containing the luciferase gene and a suitable promoter is transfected into a mammalian cell line stably expressing a receptor of interest. The plasmid used in this study contains the luciferase gene preceeded by 16 CRE units.235-238 When a ligand is added to the cell culture which activates the receptor, second messenger systems in the cell st imulate the promoter element on the plasmid, causing transcription of the lucife rase gene. The mRNA is translat ed into the luciferase protein. The amount of luciferase produced is proportional to the level of stimulation of the receptor by the ligand. Cell lysates are then supp lemented with luciferin, ATP, Mg2+ and oxygen, and the light intensity measured using a lu minometer. The intensity of light can be analyzed, leading to a

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117 quantitative measurement of the am ount of luciferase produced a nd therefore the activity of the ligand at the receptor. Functional Assay: cAMP Assay The cAMP assay is a radioassay which measur es the amount of cAMP produced in a cell. cAMP is a second messenger produced when G prot ein is activated by binding of a ligand to a GPCR, causing activation of adenylate cyclase and conversion of ATP to cAMP. The cAMP then binds to CREB which binds to promoter CRE units on DNA, stimulating transcription of certain genes. This assay differs from reporter gene assays in an important way. Reporter gene assays measure a protein produced from activatio n of the CRE-coupled genes. The cAMP assay directly measures the amount of cAMP produc ed from stimulation of the receptor. After receptor stimulation, cell lysates are isolated and tritiated cAMP (3H-cAMP) and a cAMP binding protein are incubated with the lysates. The cAMP and 3H-cAMP compete for binding to the binding protein. A st andard curve with known concen trations of cAMP incubated with a constant concentration of 3H-cAMP is also made and the unknowns compared with the standards to determine the amount of cAMP in the unknown samples. High levels of unlabeled cAMP will result in a greater amount of unlabeled cAMP bound to the binding protein, and thus lower levels of radioactivity det ected in a scintillation counter. Lo w levels of cAMP will result in a greater amount of bound 3H-cAMP and a higher scintillation counter reading. These data may be used to determine the amount of cAMP produ ced across several concentrations of ligand and may be used to plot a dose -response curve and determine EC50 values of the ligand. Though a very reliable assay, the cAMP assay ha s several drawbacks. First, it requires the use of radioactive substrates a nd specialized training and certif ication is required. Second, it is quite expensive and generally used only when more cost effective options are exhausted.

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118 Additionally, the assay is labor-intensive and not compatible with high-throughput operations. A table comparing the luciferase assay and the cAMP assay is shown in Table 4-2. Table 4-2. Comparison of luciferase and cAMP assays. Luciferase Assay cAMP Assay Detection Luminescence Radioactivity High-Throughput? Yes No Cost $$ $$$$ Ease of Assay Easy Moderate Special Equipment? Yes Yes Certification Required? No Yes Experimental Methods Cell Culture and Transfection HEK293 cells were grown in media consisti ng of DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 50 mL NCS (Newborn Calf Serum) and 5 mL Penicillin/Streptomycin. Cells were split by a dding 1 mL of a 0.1% trypsin solution and incubating for two minutes. The trypsin was then diluted with 5 mL of media and the cells dislodged by gentle pipetting. The cells were ali quotted to new plates an d 10 mL of media added. Cells were grown at 37oC and 5% CO2. OS3 cells were grown in media consisting of F10 Media supplemented with 75 mL horse serum, 12 mL FBS (Fetal Bovine Serum), 5 mL Penicillin/Str eptomycin, and 84 mg G418. Cells were split in the same way except OS3 cells must be removed from the plate with a cell scraper as they adhere more strongly to the plate surface. Transfection of HEK293 cells was perfor med using the calcium phosphate method in which 5-10 L of DNA was combined with CaCl2 and water in a 1:100 ra tio to make 500 L of solution. 2X-BBS (500 L) was added and the mi xture incubated at room temperature for 15 minutes before being added to ce lls growing on a 10cm plate. Cells were incubated overnight at

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119 35oC and 3% CO2. For stable transfection, media was changed to G418 media (regular growth media supplemented with 500 mg G418) and the me dia changed every few days until a stable population of cells began to grow. Transfection of OS3 cells was accomplished usin g Fugene 6 Transfection reagent (Roche). DNA and Fugene were combined in a 3:1 ratio. F ugene and serum-free media were combined for a total volume of 500 L and incubated at room temperature for 5 min. DNA was then added and incubated at room temperature before being adde d to cells grown in a 10cm dish. Incubation and stable transfection were accomplished in the same manner as HEK293 cells. Site-Directed Receptor Mutagenesis Insertion of Flag tag. The Flag tag was inserted on the 5’ end of the receptor gene as follows. Primers were designed which incorporat ed the Kozak sequence, start codon, and Flag tag (see chapter 7). DNA was combined with the primers, dNTPs, Taq polymerase, buffer and PCR Taq Master Mix (Brinkman Inst, Inc) The PCR reaction was run as follows: 1. 95oC, 2 min 2. 95oC, 30 sec 3. 56oC, 1 min 4. 72oC, 2 min 5. Go to 2 for 34 repititions 6. 72oC, 10 min 7. Hold 4oC Following PCR, the reaction was run on agaros e gel and the amplified fragment excised and ligated into pBKS. The plasmid was transformed into DH5 cells, and grown in a Miniprep to isolate DNA. The DNA was sequenced to confirm the presence of the Flag tag. Mutant PCR. Mutant PCR was performed as follows. Template DNA, dNTPs, Pfu polymerase (Stratagene), primers containing the desired mutation, and buffer were subjected to the following PCR reaction: 1. 95oC, 30 sec 2. 95oC, 30 sec

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120 3. 55oC, 1 min 4. 68oC, 9 min 5. Go to 2 for 12 repititions 6. Hold 4oC Following PCR, the reaction mixture was pur ified by PCR Purification (Qiagen) and incubated with Dpn1 overnight to digest temp late DNA. The DNA was then transformed into DH5 cells and the plasmid isolated by Minipre p. The sequence was confirmed and the mutant receptor excised and ligated into pcDNA3 for transfection. Transformation. Chemically competent DH5 cells (100 L) were gently mixed with 120 L of DNA and incubated on ice for 20-30 minutes The bacteria were then heat shocked for 40-60 seconds at 42oC and then incubated on ice for 2 mi nutes. SOC media (150 L) (tryptone, yeast extract, NaCl, KCl, MgCl2, MgSO4, glucose and water) was added to the tubes and then gently shaken at 210 rpm and 37oC for at least one hour. The bacter ia were then spread onto LBagar plates (tryptone, NaCl yeast extract, agar and wate r) supplemented with 100 g/L ampicillin and grown in a 37oC incubator overnight. Miniprep. Procedure was taken from Qiagen Ha ndbook. Bacterial cultures were prepared by picking a single clone from the LB-agar plat es and growing overnight in 5 mL LB broth (tryptone, NaCl, yeast extract and water) s upplemented with 100 g/L ampicillin at 275 rpm and 37oC. The bacteria were harvested by centrifuging 1.5 mL of culture in a microcentrifuge for 8 min at 6000 rpm and discarding growth medium This procedure was repeated twice and the bacterial pellet resuspended in Buffer P1. Lysis buffer P2 was added and the tubes gently mixed and held for two minutes. Neutralization Buffer P3 was then added and the tubes inverted several times to mix. The mixture was centrifuged for 10 min at 14000 rpm and the supernatant applied to a Qiagen spin column and centrifuged for 1 min at 14000 rpm. The fl owthrough was discarded and 750 L wash buffer added to the column. Th e column was centrifuged twice for 1 min each

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121 at 14000 rpm, then 40 L of water was then ad ded and centrifuged for 1 min at 14000 rpm to collect the DNA in a fresh 1.5 mL Eppendorf tube. Maxiprep. A bacterial culture was grown overni ght in 500 mL LB broth supplemented with 100 g/L ampicillin. The bacteria was ha rvested by centrifugation for 10 min at 6000 rpm and 4oC and the growth medium discarded. The pelle t was resuspended in 10 mL Buffer P1 and then lysed for 5 min in 10 mL of Buffer P2. Neut ralization Buffer P3 (10 mL) was added and the suspension incubated on ice for 20 min. The supe rnatant was obtained by centrifuging for 30 min at 14000 rpm and 4oC. The supernatant was applied to an equilibrated Qiagen column and allowed to drain by gravity. The column was washed twice with 30 mL each of wash buffer and then the DNA was eluted with 15 mL of Buffer QF. DNA was precipitated with 1.7 volumes of isopropanol and centrifuged for 30 min at 8800 rpm and 4oC. The solution was decanted and the DNA pellet collected using 70% ethanol in water. The DNA was centrifuged in a microcentrifuge for 10 min at 14000 rpm and 4oC, then the solution was decanted and the DNA air dried for 10 min. The pellet is resuspended in 100 L of TE Light buffer (Tris and EDTA, pH=8.0). Following DNA concentration determina tion, the DNA was diluted to 1 g/L with TE Light buffer. Plasmid sequencing. Plasmid sequencing using th e BDT sequencing method was accomplished as follows. DNA, a suitable sequencing primer, BDT buffer, and BDT reaction mixture were combined and run in a PCR reaction as follows: 1. 96oC, 10 sec 2. 50oC, 5 sec 3. 60oC, 4 min 4. Go to 1 for 24 repititions 5. Hold 4oC Following PCR, the reaction was purified by et hanol precipitation, dried, and submitted for sequencing.

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122 Real Time Polymerase Ch ain Reaction (RT-PCR) Stimulation. For HEK cells: 1.8 mL media (1960 L DMEM, 20 L IBMX (100x), 20 L 1% BSA in PBS) was added to a 10 cm plate (c ell confluency 90-100%). ACTH (1-24) (200L, 10-7 M) was added, and cells were incubated at 37oC for 6 hours. For OS3 cells: 1.8 mL media (1980 L OS3 me dia, 20 L IBMX (100x)) was added to a 10 cm plate (cell confluency 90100%). ACTH (1-24) (200L, 10-7 M) was added, and cells were incubated at 37oC for 6 hours. Isolation of RNA. RNA was isolated using the RN easy Mini Kit (Qiagen Sciences, Maryland, USA). Growth media was aspirated fro m cells and 600 L Buffer RLT (6 mL RLT (Qiagen Sciences, Maryland, USA) + 60 L -mercaptoethanol) was added. The cells were transferred to a 1.5 mL Eppendorf tube and homogenized by passing the lysate through a 20gauge needle ten times. One volume of ethanol was added to the homogenized lysate and mixed. The sample was placed on an RNeasy mini column (Qiagen Sciences, Maryland, USA) and centrifuged for 15 sec at 10,000 rpm. Flowthr ough was discarded, and 700 L Buffer RWI (Qiagen Sciences, Maryland, USA) was added to the column and centrifuged for 15s at 10,000 rpm. The flowthrough was discarded and 500 L of Buffer RPE (Qiagen Sciences, Maryland, USA) was added and centrifuged for 15 s at 10,0 00 rpm to dry the column. The flowthrough was discarded and 500 L of Buff er RPE (Qiagen Sciences, Ma ryland, USA) was added and centrifuged 1 min at 10,000 rpm. To elute the RNA, 40 L of RNase-free water was placed on the silica-gel membrane and centr ifuged 1 min at 10,000 rpm. The elut ion step was repeated with 40 L of RNase-free water. RNA concentration was determined by adding 4 L RNA to 796 L ddH2O and the OD260 was measured with a spectrophotomet er. The concentration was calculated from the absorbance using the equation C=OD260 x (VT/4) x 40 where C = concentration, and VT = total volume. RNA quality was determined by gel electrophoresis on a 1% agarose gel.

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123 Synthesis of cDNA. All cDNA reagents purchased from Applied Biosystems (Foster City, CA). To an optical PCR tube was added 10 L 10x Reverse Transcription Buffer, 4 L 25x dNTPs, 10 L 10x random primers, 5 L MultiScribe Reverse Transcriptase (50 U/L), 50 L TaqMan 2x Universal PCR Mast er Mix, and 71 L RNA + ddH2O (10 g RNA was calculated from measured RNA concentration). The PCR tube s were placed in an Eppendorf Mastercycler Gradient PCR machine, program: 10 min at 25oC, 120 min at 37oC. Real Time PCR. To set up the RT-PCR experiment, cDNA was first diluted to 20 ng/L (20 L cDNA + 80 L ddH2O). For 18S ribosomal cDNA det ection, the 20 ng/L cDNA was further diluted to 1 ng/2.5 L (2 L 20 ng/L cDNA + 98 L ddH2O). Assay media was prepared as follows: for each gene to be detecte d, 1.25 L of RT-PCR Assay on Demand (Applied Biosystems, Foster City, CA) was added to a tube followed by 6.25 L ddH2O and 12.5 L TaqMan 2x Universal PCR Master Mix (Applie d Biosystems, Foster City, CA). The 18S ribosomal assay media was prepared using reag ents from the TaqMan Ribosomal RNA Control Reagent Kit (Applied Biosystems, Foster City, CA). For each well, 9.625 L ddH2O, 0.125 L forward primer, 0.125 L reverse primer, 0.125 L probe, and 12.5 L of TaqMan 2x Universal PCR Master Mix (Applied Biosystems, Foster City, CA) were mixed. In a 96-well optical reaction plate, 20 L assay and 5 L 20 ng/L cDNA were added to each well for unknown gene detection, and 22.5 L assay and 2.5 L 1 ng/2.5 L cDNA was added to separate wells for 18S ribosomal detection. For each assay type, a no c DNA sample was tested, using 20 L assay and 5 L ddH2O. The plate was covered with an optical pl astic cover, centrifuged, and placed in an ABI Prism 7000 Sequence Detection System RT -PCR machine (Applied Biosystems, Foster City, CA), program: 2 min 50oC (1x), 10 min at 95oC (1x), 15 s at 95oC then 1 min at 60oC (40x).

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124 Fluorescence-Activated Cell Sorting (FACS) OS3 and HEK293 cells stably expressing the Flag-hMC2R gene were grown to 80-90% confluency. The cells were washed with 2 mL DPBS, then released from the plate by 5 min incubation at 4oC in 2 mL dissociation buffer (1x PBS, 0.02% EDTA). The cells were transferred to a 50 mL conical tube and centrifuged at 600xg fo r 5 min at room temperature. The supernatant was aspirated and cells were resuspended in 1 mL FACS buffer (1% RIA grade bovine serum albumin, 0.1% sodium azide, and 1x PBS pH 7.2). The cell suspension (200 L) was aliquoted into each of four FACS tubes, labeled as such: no label, isot ype, surface, and total. Mouse IgG (1.5 L) was added to each tube to block Fc receptors, and incubated 10 min at room temperature. To the tube containing the isot ype control, 1 L APC-labeled mouse IgG1 k1 isotype control was added. To the surface and to tal tubes, 1 L anti-Flag-APC was added. All tubes were incubated at room temperature for 45 min. After incubation, 4% formaldehyde in 1x PBS was added to each tube for a final concentra tion of 2% (v/v). Tubes were held at room temperature for 15 min, then centrifuged at 600xg for 5 min at room temperature. The supernatant was decanted, and 1 mL saponin bu ffer (1% RIA grade bovine serum albumin, 0.1% sodium azide, 0.5% saponin, 1x PBS pH 7.2) was a dded to each tube and incubated 20 min. The tubes were centrifuged at 600xg fo r 5 min at room temperature a nd the supernatant decanted. To the isotype control tube 1 L of APC-labeled mouse IgG1 k1 isotype control was added and to the tube for total expression 1L anti-Flag-APC was added then incubated at room temperature for 1 hour. All tubes were washed three times with saponin buffer as follows: 1 mL saponin buffer was added then the tubes were centrifuged at 600xg for 5 min at room temperature nad the supernatant was decanted. Tubes were held at 4oC until analyzed. Flow cytometry was performed on a FACSCalibur by Becton Dickin son. Cells were prepared for deconvolution microscopy by adding 1 mL of DAPI stain to all tubes, then cytospinning the samples at 600xg

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125 for 4 min at room temperature. Slides were washed in 1x PBS, air dried, and mounted with Biomedia Gel Mount. Slides were stored at 4oC until analyzed. Luciferase Reporter Gene Assay Pharmacological analysis was performed by luci ferase reporter gene assay. On the day of transfection 96-well black-wall clear-bottom luminescence plates were treated with 100 L working collagen solution per well. HEK-293 cells stably expressing the melanocortin receptors were transiently transfected with CRE/luciferase reporter gene and 0.5 g of GFP plasmid per cell type to test transfection efficiency. Twenty-four hours post -transfection the plates were washed with 200 L DMEM per well and transfecte d cells were plated at 100 L per well. Fortyeight hours post-transfection the cells were stimulated with 100 L peptide (10-4 10-10 M) or forskolin (10-4 M) control in assay medium (DME M containing 0.1 mg/mL BSA and 0.1 mM IBMX) for 6 h then the cells were lysed usi ng lysis buffer (0.1M potassium phosphate buffer pH=7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA). Protein analysis was performed by adding 10 L of cell lysate to 200 L protein dy e in 96-well assay plat es. Protein content was measured at OD595 using a 96-well plate reader (Molecu lar Devices). To the remaining cell lysate 100 L of luciferase assay buffer (30 mM tricine, 2 mM ATP, 15 mM MgSO4, 10 mM DTT) and 100 L 1mM D-luciferin were a dded. Luminescence was measured using a luminometer. Data points were normalized both to the relative protein content and non-receptor dependent forskolin stimulation. Functional Assay: cAMP Assay cAMP TRK432 kit purchased from Amersham Biosciences. EBSS Assay buffer (1 mL of 1 M HEPES pH 7.4, 1 mL 200 mM Gl utamine, 6.3 mL 17.5 g NaHCO3/500 mL stock, 100 mg bovine serum albumin, 91.7 mL 1X EBSS) was prepared first and then 1X IBMX in EBSS buffer was made for compound dilutions (1 mL 100x IBMX per 100 mL EBSS assay buffer).

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126 Compound dilutions of ACTH (1-24) 10-6-10-11 M were prepared in assay buffer containing IBMX. Growth media was aspira ted from the cells and cells were washed with EBSS assay buffer. Cells were incubated for 30 min at 37oC in 500 L EBSS assay buffer. After incubation, 500 L of compound dilution were added per well and cells incubated for 6 h at 37oC. Cells were lysed cells with 1 mL 100% ethanol and tran sfered to labeled 1.5 mL Eppendorf tubes which were placed on ice for 30 min. Tubes were centrifuged at 4oC for 10 min at 1900 g and the supernatant transferred to labeled 12x75mm gla ss tubes. The supernatant was evaporated at 55oC in a water bath under a stream of nitrogen ga s until dry. cAMP standards were prepared (16 pmol/mL 1 pmol/mL). The dry supernatant was resuspended in 150 L Tris EDTA buffer (50 mM Tris/HCl, 4 mM EDTA pH 7.5 ), and vortexed to mix. An aliquot of each sample (50 L) was transferred to labele d 1.5 mL Eppendorf tubes. 3H-cAMP tracer (50 L) and 100 L binding protein was added to each tube vortexed, and incubated at 4oC for 2 h up to overnight. Charcoal suspension (100 L) was added to all tubes, vortexed, and centrifuged for 5 min at maximum speed. The supernatant from each tube (200 L) was added to a separate scin tillation vial filled with 5 mL ScintiVerse scintillation fluid. Vials were counted using a scintillation counter for 1 min per sample. Data Analysis Real Time PCR, luciferase, and cAMP data analysis was performed using Microsoft Excel and Graphpad Prism 4.0. FACS analysis was perfo rmed using CellQuest as well as Microsoft Excel and Graphpad Prism 4.0. All experiments were performed a minimum of three times.

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127 CHAPTER 5 STRUCTURE-ACTIVITY RELATIONSHIP ST UDIES OF PEPTIDES: A STUDY OF INTRAMOLECULAR CATIONINTERACTIONS IN MELA NOCORTIN AGONISTS AND THE EFFECTS ON AGONIST AN D ANTAGONIST SELECTIVITY Peptides in this study (peptides 21-32) we re synthesized, purified and analytically characterized by Krista R. Wilson. Pharmacology was performed by Michael S. Wood, Nicholas B. Sorenson, Dong V. Phan, and Zhimin Xiang of the same laboratory. Data Analysis was performed by Carrie Haskell-Luevano and Krista R. Wilson. Introduction Noncovalent interactions play an important role in stabilization of the secondary and tertiary structure of proteins and peptides as well as in protein-prot ein, ligand-receptor, and DNA-protein interactions.239,240 Over the past decade it has beco me clear that the electrostatic interaction between a cation and the negative potential f ound on the face of a system may be significantly involved in ligand -receptor interactions as we ll as in peptide and protein folding.239,240 Figure 5-1. Cationinteraction between the positive charge of arginine and the negative potential on the face of th e Phe aromatic system. Many factors are involved in shaping a pol ypeptide chain into a bioactive molecule. Secondary and tertiary structural determinations are driven by noncovalent interactions between backbone and side chain molecular elements as well as interaction with solvent and other proteins in the environment.239,240 These noncovalent interactions can be quite strong and involve

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128 forces such as hydrogen bonding, electrostatic (ionic) inter actions, hydrophobic attraction, dispersion forces, aromatic interactions, dipole-dipole in teractions, and dipole-quadropole interactions just to name a few.239,240 One noncovalent force that has recently been identified as having a significant impact on protein and peptide structure is the cationinteraction.239,240 In general, the cationinteraction is the association of a ca tion or partial positive charge with the negatively charged face of an aromatic system (Figure 5-1). In prot eins, these interactions can be quite complex, and have been s hown to be involved in secondary -helix, -sheet and turn stabilization, protein and peptide folding, liga nd-receptor binding and signaling, protein-protein interaction, DNA-protein interactions and antibody-antigen recognition.239,240 In native proteins, cationinteractions occur between specific amino aci d side chains. The cation of Arg, Lys, and His may interact with the aromatic systems of Trp, Tyr, and Phe (Figure 5-2).239,240 Figure 5-2. Structure of natural and unna tural amino acids used in this study.

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129 Benzene is the prototypic example of a simple ar omatic system, and is used here as a basis for understanding the aromatic systems of Trp, Ty r, and Phe. For general purposes, benzene is often considered a nonpolar molecule (Figur e 5-3A). Though it does not exhibit a permanent dipole moment, this does not mean that the mol ecule does not contain a charge distribution. The orbitals of the aromatic system are arranged in such a manner as to produce six dipole moments, which give benzene a permanent, s ubstantial quadrupole moment (-28.3 kcal/mol 1.2) (Figure 5-3B,C).241 This results in a significant charge distribution where the interior of the aromatic ring contains a negative charge and th e outer ring is positively charged (Figure 5-3D). The negatively charged face can then interact with surrounding positive charges in a manner similar to the electrostatic interaction of two ions. Figure 5-3. Benzene. A. Structure of benzene. B. Pi orbitals of benzene from a side view. C. Schematic of the quadropole moment of benzen e, side view. D. Charge distribution of benzene; blue indicates a negative ch arge, red indicates a positive charge. It has been known for some time that small metal cations were able to bind strongly to benzene, with smaller atoms binding stronger than larger atoms.242 It was therefore postulated that this phenomenon could be applied to system s with partial positive charges and to organic cations such as the ammonium ion.243,244 In fact, it has been shown that aromatic systems can

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130 interact with both full and part ial charges, such as those f ound in highly polarized molecules such as water and amines.243,244 In peptides and proteins this results in a significant amount of stabilization obtained from the interaction of cati onic amino acid side chains (ie. arginine, lysine, histidine) with aromatic amino acid side chai ns (ie. phenylalanine, tyrosine, tryptophan).243,244 As Arg, Lys, and His are ionized at physiological pH, they may interact with the aromatic systems in a cationinteraction. Though these interactions were first observed in the gas phase, studies by the Dougherty laboratory245 designed artificial receptor system s that were able to exhibit cationinteractions in an aqueous environment.245 In fact, the aromatic “receptors” were able to pull cations out of water and bind them in an aromatic environment.245 This is significant as noncovalent interactions must compete with the solvation energy of the positive charge interacting with the polar groups of water. To bind the aromatic system, the cation must undergo a “desolvation penalty”246-248 to break its aqueous interactions and bi nd to the aromatic. This change in energy due to desolvation has been used to describe the differing binding stre ngths of arginine and lysine. In general, arginine associates more strongly with aromatic systems than lysine.243,244 The guandinyl group is chemically different from a primary amine and thus associates differently with aromatic systems. Interestingly, in the ga s phase the ammonium ion interacts more strongly than the guanidinium ion.249 This may be related to the small size of the ammonium ion (smaller ions associate more strongly than larger ones) and the charge distri bution on the guanidinium ion. Since arginine is much more likely to be involved in a cationinteraction, there must be factors other than electrostatic effects involve d in these interactions One theory relates desolvation energy to strength of interaction (Figure 5-4).250 The guanidinium ion has two

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131 potential sites for ionization (Fig ure 5-4A). This decreases the desolvation penalty because one ion can interact with the aromatic system and th e other cation can maintain interactions with the aqueous environment, lowering th e energy related to desolvation.250 The ammonium ion only has one site for ionization and thus must relinquish its interactions with water to bind the aromatic system (Figure 5-4B). This results in a much greater desolvation penalty associated with lysine binding to aromatics than arginine.250 This desolvation penalty may be reduced in lysine due to the partial positive charge associated with the carbon. This carbon is highly polarized and maintains a partial positive charge that can intera ct with either an aromatic or water. This interaction is not as strong as a full cati on, but may decrease the desolvation penalty.251 These observations may be used to explain the differenc es associated with arginine versus lysine selectivity. Figure 5-4. Cationinteractions of Arg and Lys with Phe and water. A. and B. show two different conformations by which Arg may interact with Phe. C. and D. show conformations of interac tion between Lys and Phe. The three most common hydrophobic amino acids involved in cationinteractions are phenylalanine, tyrosine, and tryptophan.243,244 In the gas phase, an indo le moiety binds cations much stronger than e ither phenol or benzene.239,252,253 In protein database searches, 26% of all

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132 tryptophan residues are located near cations.251 This is in agreement with the observation that tryptophan is the aromatic amino acid mo st likely to be involved in cationinteractions as it has the greatest negative potential. The side chains of phenylalanine and tyrosine have similar binding properties;253 however, if the hydroxyl of tyrosine is hydrogen bonded, or if the negative potential of the oxygen interacts directly with the cation, the binding prop erties of tyrosine increase as compared to phenylalanine.251 Figure 5-5. Structure and pharmacology of the synthe tic melanocortin agonist MTII (left) and the synthetic melanocortin antagonist SHU9119 (ri ght). The lactam bridge is shown in blue and the differing aromatic systems [M TII = Phe; SHU9119 = DNal(2’)] are in red. EC50 values in nM are shown below the st ructures along with associated errors. Antagonist pA2 values are shown for SHU9119 at the mMC3R and mMC4R.254 All known endogenous melanocortin peptides cont ain the same core tetrapeptide sequence, His-Phe-Arg-Trp. These four residues are esse ntial for potent activity at the melanocortin receptors.115 SAR studies of -MSH have shown that the mi nimal essential sequence for stimulation is Ac-Phe-Arg-Trp-NH2, though addition of His results in a significant increase in ligand potency.114,115 These SAR studies resulted in the di scovery of NDP-MSH, a derivative of -MSH in which the Met was replaced with a Nle to prevent oxidation and the Phe is replaced

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133 with a DPhe to significa ntly increase potency.4 This molecule was much more potent than the endogenous peptide. Further cyclization and de letion studies of NDP-MSH resulted in the development of MTII (Ac-Nle-[Asp-His-DPhe-Arg-Trp-Lys]-NH2) (Figure 5-5), a small cyclic agonist that is more potent than either -MSH or NDP-MSH.117,118,255 During the course of this investigation, it was discovered that replacement of DPhe wi th a DNal(2’) resulted in a compound (SHU9119, Figure 5-5) (Ac-Nle-[Asp-His-DNal(2’)-Arg-Trp-Lys]-NH2) with potent antagonist activity at the mMC3 and mMC4R.117,118,255 It is hypothesized that there may be a cationinteraction between the arginine and the adjacent aromatic residue and that the differences between the interacti on with DPhe and DNal(2’) and ar ginine may have an effect on agonist and antagonist selectivity.43 Background Pharmacology Holder et al. synthesized a number of single-subs titution derivatives of the core tetrapeptide sequence His-Phe-ArgTrp in order to investigate the ideal structural features for melanocortin activity.6-9 They systematically replaced each amino acid with a number of natural and unnatural amino acids. During the course of these studies, it was observed that the Phe and Arg positions had very specific requirements for activity, and that the Phe position seems to be significantly involved in agoni st versus antagonist activity. Some significant compounds synthesized during this st udy are shown in Table 5-1.

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134Table 5-1. SAR Studies of Select ed Melanocortin Tetrapeptides116-14;6-9 and Pharmacology Data of Arg Substitutions 256-20 mMC1R mMC3R mMC4R mMC5R # Structure EC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM) 1 Ac-His-Phe-Arg-Trp-NH2 76903590 4370535 2110243 10328 2 Ac-His-DPhe-Arg-Trp-NH2 20.10.57 1569.2 17.22.78 3.960.94 3 Ac-HisAla -Arg-Trp-NH2 300006100 >100000 >100000 >100000 4 Ac-His -(pI)DPhe -Arg-Trp-NH2 60.413.4 PA pA2 = 7.250.18 25.09.78 1.600.35 5 Ac-HisNal(1’) -Arg-Trp-NH2 152003100 >100000 >100000 112002300 6 Ac-HisDNal(1’) -Arg-Trp-NH2 35664 41001000 30365 51.44.07 7 Ac-HisNal(2’) -Arg-Trp-NH2 220005600 >100000 >100000 74001800 8 Ac-His -DNal(2’) -Arg-Trp-NH2 16741 PA pA2 = 6.530.09 pA2 = 7.780.18 34.76.75 9 Ac-His-DPheAla -Trp-NH2 34001600 420008900 230004100 69001500 10 Ac-His-DPheLys -Trp-NH2 770500 255001900 830140 32014 11 Ac-His-DPheOrn -Trp-NH2 61002200 556002800 65001900 54030 12 Ac-His-DPheDap -Trp-NH2 93003100 389005800 3800893 61040 13 Ac-His-DPheAsp -Trp-NH2 434009700 >100000 >100000 164007900 14 Ac-His-DPhe -Glu -Trp-NH2 429009700 654009100 155002200 2000970 15 Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 0.020.002 0.140.016 0.040.007 0.270.03 16 Ac-Nle-c[Asp-His-Phe-Arg-Trp-Lys]-NH2 23.212.8 1.730.12 457.5 1.621.0 17 Ac-Nle-c[Asp-His-DNal(2’)-Arg-Trp-Lys]-NH2 0.200.08 pA2 = 9.540.23 50%@10M pA2 = 10.140.20 1.790.65 18 Ac-Nle-c[Asp-His-Nal(2’)-Arg-Trp-Lys]-NH2 12232 21.94.8 80%@10M 817.2 60%@10M 1.850.60 19 Ac-Nle-c[Asp-His-DNal(1’)-Arg-Trp-Lys]-NH2 0.680.39 1.230.27 0.380.07 70%@10M 3.20.7 20 Ac-Nle-c[Asp-His-Nal(1’)-Arg-Trp-Lys]-NH2 380150 246170 85%@10M 14227 11.18.0 Compounds 15-20 were synthesized in the Haskell-Luevano laboratory at the University of Florida. Compounds 15,18,19 and 20 were synthesized by J. Ryan Holder, Compound 16 was synthesized by Jay W. Schaub, and Compound 17 was synthesized by Fernanda Marques. Indicated errors rep resent the standard error of the mean for at least three independent experiments. >100000 indicates that no agonist activity was observed at agonis t concentrations up to 100M concentrations. PA = partial agonist. Compounds not possessing full agonist activity were assayed for antagonist activity using the Schild analysis against the agonist MTII.

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135 SAR at the Phe position7 revealed that similar to previous studies, replacing the LPhe with a DPhe resulted in a compound that is >100-fo ld more potent at the mMC4R. Additionally, replacement with an Ala significantly decrea sed the agonist potency at the mMC1R, and completely abolished all activity at the mMC3-5R, indicating that the aromatic ring is important for activity. Replacing the single aromatic ring of Phe with either a double aromatic system (napthylalanine [Nal]) or with a para-substituted phenyl ring gave interest ing results. When Phe was replaced with either a (pI)DPhe or a DNal( 2’) residue the tetrapeptide showed antagonist activity.7 The (pI)DPhe compound showed mixed pharm acology with both partial agonist and antagonist activity at the mMC3R and full agoni st activity at the mMC4R, and the DNal(2’) compound was an antagonist at both the mMC3R and the mMC4R, as well as a partial agonist at the mMC3R.7 Table 5-2. Antagonists of the MC3R and MC4R containing DNal(2’). Compound Name Sequence Reference PG-914 Ac-Nle-c[Asp-Che-DNal(2’)-Arg-Trp-Lys]-NH2 257 SHU9119 Ac-Nle-c[Asp-HisDNal(2’)-Arg-Trp-Lys]-NH2 117,255,258 HS014 c[Cys-Glu-His-DNal(2’)-Arg -Trp-Gly]-Pro-Pro-Lys-Asp-NH2 259 HS024 c[Cys-Nle-Arg-His-DNal(2’)-Arg-Trp-Gly-Cys] 260 HS9510 c[Cys-Glu-His-DNal(2’)-Arg-Trp-Cys] 261 The effects of substituting a DNal(2’) for DPhe have been well characterized. Its effect on melanocortin potency was observed during SAR studies of MTII, which resulted in the discovery of the potent mMC3R and mMC4R antagonist SHU9119.117,255,258,262,263 SHU9119 is an agonist at the mMC1,5R and a partial agonist at the mMC3R, but is a potent antagonist at the mMC3,4R.117,255,258,262,263 Table 5-2 depicts several peptid es containing the DNal(2’) moiety which are antagonists at the MC3R and MC4R.117,255,257-259,261,263,264 A plethora of SAR studies have been performed on SHU9119 which consistent ly demonstrate that th e substitution of DPhe

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136 with a DNal(2’) results in a compound with se lective antagonist activity at the MC3R and MC4R.257,259-261,265-273 For LNal(2’) or either the Dor Lform s of Nal(1’), there was no antagonist activity observed, and the compounds were much less po tent than the DPhe or DNal(2’) compounds. This data, along with data from MTII and SHU9 119, has lead to the hypo thesis that the Phe position may be involved in the determination of agonist vs antagonist potency at the mMC3R and mMC4R. Tetrapeptide SAR studies at the Arg position9,274,275 revealed that the cationic character of the guanidine side chain is important for agoni st activity. When Arg was replaced with an Ala there was a significant d ecrease in agonist activity observe d at all melanocortin receptors.9,274,275 Additionally, decreasing the cationic characte r with a Lys resulted in a compound with intermediate potency (less than Arg, more than Ala). Decreasing the chain length beginning with Lys (4C), then Orn (3C), and finally Dap (2C) resulted in a stepwise decrease in potency. Furthermore, replacement with an anionic amino acid such as Asp and Glu resulted in either a complete loss of activity (Asp) or a significant decr ease in activity (Glu). This data indicates that there may be an anionic binding pocket that requ ires a cation in the lig and for efficient binding. Data from these studies were used in the de sign of compounds in the present work. In this study, MTII,117,258 a cyclic synthetic melanocortin peptide, was modified at the arginine and Dphenylalanine positions to i nvestigate the role cationinteractions may play in the agonist or antagonist potency of this ligand at the melanoc ortin receptors. It is hypothesized that when combined with any one of six aromatic systems [Phe, DPhe, Nal(1’), DNal(1’), Nal(2’), or DNal(2’)], peptides with an arginine should e xhibit the greatest potency and those with an alanine should have a significant loss of activity, with lysine peptides exhibiting an intermediate

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137 potency. Additionally, in accordance with th e melanocortin antagonist SHU9119, compounds containing a DNal(2’) should exhi bit antagonist activity. Napthylal anine derivatives are used because the double aromatic ring results in a grea ter negative electrostatic potential than the single ring of phenylalanine, and th us should interact more strongl y with the cation (Figure 5-6). Figure 5-6. Schematic of a cationinteraction (dotted lines) betw een the cationic side chain of Arg (blue) and the nega tive potential of the systems of DPhe and DNal(2’) (red). Results Peptide synthesis. All peptides were synthesized using standard Boc chemistry in a manual reaction vessel (Peptides International, L ouisville, KY). The peptides were purified to homogeneity by semi-preparative reversed-phase high-performance liquid chromatography (RPHPLC). Peptide purity was determined using ma ss spectrometry and analytical RP-HPLC in two solvent systems (Table 5-3)

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138 Table 5-3. Analytical Data for Pe ptides Synthesized in the CationStudy # HPLC k’ (System 1) HPLC k’ (System 2) % Yield m/z (M, calcd) m/z (M+1, exptl) 21 6.22 11.13 >95% 995.5 996.8 22 6.70 10.88 >95% 938.5 939.7 23 6.20 10.96 >98% 995.5 997.3 24 5.71 11.35 >98% 938.5 939.9 25 7.17 12.47 >95% 1046.2 1047.0 26 6.81 12.06 >98% 989.1 990.9 27 6.71 11.09 >95% 1046.2 147.5 28 6.61 12.76 >98% 989.1 990.8 29 6.98 12.41 >95% 1046.2 1047.7 30 6.80 13.16 >98% 989.1 989.9 31 6.59 11.54 >98% 1046.2 1046.8 32 6.63 12.96 >98% 989.1 989.8 HPLC k’ = [(peptide retention time – solvent retention time)/(solvent retention time)] in solvent system 1 (10 % acetonitrile in 0.1 % trifluoroacetic acid/water and a gradient to 90 % acetonitrile over 35 min) or solvent system 2 (10 % methanol in 0.1 % trifluoroacetic Acid/w ater and a gradient to 90 % methanol over 35 min). An analytical Vydac C1 8 column (Vydac 218TP104) was used with a flow rate of 1.5 ml/min. The peptide purity was determined by HPLC at a wavelength of 214 nm by comparison of the area under the curve of compound peak compared to all other peaks.

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139Table 5-4. Pharmacology Data for Peptides in the CationStudy mMC1R mMC3R mMC4R mMC5R # Structure EC50 (nM) Fold Diff EC50 (nM) Fold Diff EC50 (nM) Fold Diff EC50 (nM) Fold Diff -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe -Arg-Trp-Gly-Lys-Pro-Val-NH2 0.70.23 2.140.46 2.560.29 2.00.20 NDPMSH Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 1.00.79 0.090.02 0.10.01 0.220.03 21 Ac-Nle-[Asp-His-DPhe-Lys-Trp-Lys]-NH2 0.130.12 6 2.290.70 16 0.660.37 17 0.860.65 3 22 Ac-Nle-[Asp-His-DPhe-Ala-Trp-Lys]-NH2 1.770.58 89 4.971.08 36 2.190.98 55 0.570.20 2 23 Ac-Nle-[Asp-His-Phe-Lys-Trp-Lys]-NH2 311106 13 7614 44 457.5 37 1.621.0 0.8 24 Ac-Nle-[Asp-His-Phe-Ala-Trp-Lys]-NH2 1400315 60 1064160 615 700260 574 19.73.56 10 25 Ac-Nle-[Asp-His-DNal(2’)-Lys-Trp-Lys]-NH2 2.040.90 10 pA2 = 8.380.1 14 pA2 = 9.030.3 13 8.982.49 5 26 Ac-Nle-[Asp-His-DNal(2’)-Ala-Trp-Lys]-NH2 8.441.7 42 pA2 = 7.440.2 125 pA2 = 8.540.4 40 2.330.80 1.3 27 Ac-Nle-[Asp-His-Nal(2’)-Lys-Trp-Lys]-NH2 1000370 8 420100 50%@10M 19 1240400 15 72.447 40 28 Ac-Nle-[Asp-His-Nal(2’)-Ala-Trp-Lys]-NH2 1820700 15 140005300 640 >100000 43057 230 29 Ac-Nle-[Asp-His-DNal(1’)-Lys-Trp-Lys]-NH2 0.980.36 1 30.916 25 4.271.43 11 3.200.7 8 30 Ac-Nle-[Asp-His-DNal(1’)-Ala-Trp-Lys]-NH2 5.382.79 8 74.822 61 44.67.1 117 7.411.1 18 31 Ac-Nle-[Asp-His-Nal(1’)-Lys-Trp-Lys]-NH2 1800700 5 4800900 20 1900800 13 255112 23 32 Ac-Nle-[Asp-His-Nal(1’)-Ala-Trp-Lys]-NH2 2600150 7 3500250 60%@10M 14 2600470 60%@10M pA2 = 6.150.2 18 3200930 288 Indicated errors represent the st andard error of the mean for at least three independent experiments. >100000 indicates that no agonist activity was observed at agonist concentrations up to 100M concen trations. PA = partial agonist. Compounds not possessing full agonist activity were as sayed for antagonist activity using the Schild analysis against the agonist MTII. Antagonist fold differences were determined using Ki values; Ki = -logpA2. For each aromatic system, the Arg compound is defined as 1, with the fold differences of Lys and Ala compounds calculated compar ed to Arg (Arg compounds in T able 5-1).

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140 Biological evaluation. Table 5-4 compares the cAMP stimulation determined by the galactosidase reporter gene assay209 of the MTII derivatives at the mMC1,3-5R with the endogenous peptide -MSH and the known synthetic agonist MTII as controls. Compounds with the Arg substitution are shown in Table 5-1. When DPhe was the aromatic system, the orde r of potency of arginine>lysine>alanine was observed at all four receptor s. At the mMC1R, the DPhe-Arg compound 15 (MTII) is a subnanomolar agonist with 0.02 nM activity. When the arginine is converted to a lysine (compound 21) there was a 6-fold decrease in potency, and when an alanine (compound 22) was inserted, there was an 89-fold decrease in poten cy as compared to compound 15. At the mMC3R, MTII is a 0.14 nM agonist. Additi on of lysine in compound 21 resu lts in a 16-fold decrease in potency and the presence of alanine in compound 22 causes a 36-fold decrease in potency. At the mMC4R, MTII is a 0.04 nM agonist, but substitution of the arginine for a lysine or an alanine results in a 17-fold and 55-fold decrease in potency, respectively. The potency trend is not observed at the mMC5R, however, as all three co mpounds are within 3-fold experimental error (error inherent in all laboratory experiments) of each other and are therefore equipotent. The values for compound 15 (MTII, DPhe-Arg), and compound 22 (DPhe-Ala) are in agreement with previous SAR studies of MTII.117,255,258 When the aromatic system is Phe, there wa s a decrease in potency observed compared to the DPhe compounds. At the mMC1R, compoun d 16 (Phe-Arg) is a 23.2 nM agonist. Compound 23 (Phe-Lys) exhibits a 13-fold decrease in potency and com pound 24 (Phe-Ala) exhibits a 60fold decrease in potency as compared to compound 16. At the mMC3R, compound 16 is a 1.73 nM agonist while compounds 23 and 24 are 44and 615-fold less potent than the arginine compound 16, respectively. At the mMC4R, com pound 16 is a 1.22 nM agonist. As before, the

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141 lysine compound 23 is 55-fold less potent and the alanine compound 24 is 574-fold less potent than the arginine compound 16. At the mMC5 R, the arginine compound 16 and the lysine compound 23 are equipotent, while the alanine compound 24 exhibits a 10-fold decrease in potency. SHU9119 is identical to MTII except that the DPhe of MTII is replaced with DNal(2’). Previous studies have shown that this com pound is a subnanomolar full agonist at the mMC1R and the mMC5R, and an antagonist and partial ag onist at the mMC3R and an antagonist at the mMC4R.117,118,255 The results reported here for com pound 17 (DNal(2’)-Arg) are similar to previously reported values.117,118,255 Compound 17 is a 0.20 nM agoni st at the mMC1R with partial agonist activity. The DNal(2’)-Lys compo und 25 exhibits a 10-fold decrease in potency compared to compound 17, and the DNal(2’)-Ala compound 26 is 42-fold less potent than compound 17 at the mMC1R. At the mMC3R, all three compounds are antagonists with partial agonist activity observed in compound 17 only. St ill the same potency trend continues, as compound 17 (Arg) is the most potent antagon ist, followed by compound 25 (Lys) and then compound 26 (Ala). This antagonist trend is the same at the mMC4R, though all three compounds are more potent antagonists than the same compound at the mMC3R. Once again, the potency trend changes for compounds tested at the mMC5R. The lysine compound 25 is 5fold less potent than the arginine compound 17, while the alanine compound 26 is equipotent with the alanine compound 17. When the aromatic system is changed from DNal(2’) to Nal(2’) th e pharmacology differs significantly. These compounds are much less potent agonists and are not an tagonists at any of the receptors. At the mMC1R, compound 18 [Nal(2 ’)-Arg] is a 122 nM agonist. When the cation is changed to a lysine in compound 27, there is an 8-fold decrease in potency, and when it is

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142 changed to an alanine in compound 28, there is a 15-fold decrease in potency. At the mMC3R, compound 18 [Nal(2’)-Arg] is a 430 nM agonist, and only exhibits partial agonist activity. Compound 27 [Nal(2’)-Lys] is 19-fold less potent than compound 18, and compound 27 [Nal(2’)-Ala] is 640-fold less potent than compou nd 18, though they are both full agonists while compound 18 is a partial agonist. At the mMC4R, the arginine compound 18 is an 81 nM partial agonist, while the lysine compound 27 is a 15-fo ld less potent full agonist. When the cation is removed by inserting an alanin e in compound 28, the compound does not exhibit any agonist or antagonist activity at the mMC4R. At the mMC5R compounds 18, 27, and 28 follow the same arginine>lysine>alanine potency trend as the ot her receptors. At the mMC5R, compound 18 is a potent 1.85 nM full agonist, while compound 27 [Nal(2’)-Lys] is 40-fold less potent and compound 28 [Nal(2’)-Ala] is 230-fold less potent than compound 18. The next aromatic system tested was DNal( 1’). At the mMC1R, compound 19 [DNal(1’)Arg] and compound 29 [DNal(1’)-Lys] were equipotent subnanomolar agonists. Compound 30 [DNal(1’)-Ala] was 8-fold less potent than the ot her compounds at the mMC1R. At the mMC3R, the arginine compound 19 is a 1.23 nM agonist. The lysine compound 29 is 25-fold less potent than compound 19 and the alanine compound 30 is 61-fold less potent than compound 19. At the mMC4R, compound 19 is a 0.38 nM agonist, com pound 29 exhibits an 11-fold decrease in potency, and compound 30 exhibits a 117-fold decr ease in potency. At the mMC5R, the potency trend is followed, as the alan ine compound 30 is 18-fold less pot ent than the arginine compound 19 while the lysine compound 29 is 8fold less potent than compound 19. The final aromatic system tested was Na l(1’). These compounds are less potent than the corresponding DNal(1’) compounds. At the mMC1 R, compound 20 [Nal(1’)-Arg] is a 380 nM agonist. The lysine compound 31 is 5-fold less potent, and the alanine compound 32 is 7-fold

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143 less potent. At the mMC3R, compound 20 is a 24 6 nM partial agonist while compound 31 is a 20-fold less potent full agonist. Compound 32, howev er, is only 14-fold less potent than the arginine compound 20, making it more potent than the lysine co mpound 31, though it is a partial agonist. The potency trend returns at the mM C4R. Compound 20 [Nal(1)-Arg] is a 142 nM agonist and the lysine compound 31 is 13-fold less potent. Surprisingly, when an alanine is included in compound 32, the compound becomes a partial agonist and an antagonist at the mMC4R. Finally, at the mMC5R, compound 20 is an 11.1 nM agonist, co mpound 31 is 23-fold less potent, and compound 32 exhibits a 288fold decrease in potency with an EC50 value of 3200 nM at the mMC5R. Discussion The interaction between a cation and an aromatic system has recently gained popularity as a significant noncova lent interaction in biological systems.239,240 Recent analysis of the Protein Databank (PDB) has indicated that there is one cationfor every 77 amino acids in proteins.251 Additionally it was observed that Arg is found in cationinteractions more often than Lys and over 70% of Arg residues are close to an aromatic.251,276 Tryptophan is the most common aromatic residue to associate w ith a cation, with over 25% of all Trp residues being located near a cation251 and 7.3% of all cationinteractions are found between adjacent amino acid residues (by X-ray crystallography) helpi ng to establish the 3D conformation of the protein or peptide.251 Cationinteractions are also commonly found in the i and i +4 positions, indicating their importance in the stabilization of the -helix.251,277 Cationinteractions have been found in many di fferent capacities. They have been shown to play a role in transcription activation,278,279 DNA binding to proteins,280 antibody-antigen interactions,281-283 and membrane protein interactions284,285 just to name a few. They have been shown to be important in the binding of ligand and receptor in acetylcholine receptors,286-292

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144 serotonin receptors,290,293,294 GABA receptors,295-298 NPY,299 angiotensin 2 receptors,300 and in antimicrobial peptide action.301 Recently, the cationinteraction has been exploited as a tool in synthetic organic chemistry as reviewed.302 In the present study, the differential cationinteractions between arginine and lysine were used to investigate the presence of a cationinteraction in the melanocortin synthetic agonist MTII. Arginine, lysine, and alanin e were coupled with six aromatic systems, Phe, DPhe, Nal(1’), DNal(1’), Nal(2’), and DNal(2’). It wa s hypothesized that if there is a cationinteraction between the Arg and DPhe of MTII, then when the Arg is replaced with Lys there will be an observable decrease in potency and when Ala is substituted there will be an even greater decrease in potency resulting from a decrease in cationinteraction strength. Additionally, as MTII (Arg-DPhe) is an agoni st, and SHU9119 [Arg-DNal(2’)] is an antagonist, it is hypothesized that the difference in the interaction between Arg and either DPhe and DNal(2’) may be responsible for agonist versus antagonist selectivity at the hMC3,4R. D-Phenylalanine The first aromatic system investigated was DPhe, which is the aromatic amino acid present in MTII.117,255,258 These peptides (peptides 15, 21 and 22) were the most potent compounds tested in this study, with all values in the subto low-nanomolar ra nge. At the mMC1,3, and 4R the reported values agree with the hypothesis that Arg > Lys > Ala. At each of these receptors, the Arg compound is significantly more potent than the Lys compound, with the Ala compound being the least potent compound. These date indicate that there may be a cationinteraction which is important for ligand activity. At th e mMC5R, however, all three compounds were within 3-fold experimental error of each other and no significant differences were observed. This may indicate that the cationinteraction is more important fo r activity at the mMC1,3, and 4R

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145 than at the mMC5R. Bar graphs showing the differ ences in potency at each receptor are shown in Figure 5-7. Figure 5-7. Bar graph of peptides 15, 21 and 22 with DPhe as the aromatic system at the mMC1,3-5R. Phenylalanine The next group of peptides (peptides 16, 23 a nd 24) contain an LPhe in place of the DPhe. These compounds differ from the DPhe compounds only in the conformation of the Phe side chain. It has already been reported that cha nging the LPhe in the endogenous melanocortin ligand -MSH to a DPhe (to make NDP-MSH) resu lts in a significant increase in potency.4 The results reported herein agree with this diffe rence in potency. As compared to the DPhe compounds, the LPhe compounds were significantly less potent. Ho wever, the same trends are observed at the mMC1,3, and 4R as before. At ea ch of these receptors, the Arg compounds were the most potent, followed by a significant loss of potency when a Lys was inserted, and an even greater loss of potency when the cation was s ubstituted with an Ala. Once again, these data support the hypothesis of a significant cationinteraction between Ar g and Phe. A different trend was observed at the mMC5R. The Arg a nd Lys compounds were equipotent, and the Ala compound was 10-fold less potent than the other tw o compounds. This data may indicate that at this receptor, the identity of the cation is less important, and potency is maintained as long as a

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146 positive charge is present. When the positive charge is completely removed, however, the potency is decreased, though not as significantly as at the ot her receptors. These data are depicted in Figure 5-8. Figure 5-8. Bar graph of peptides 16, 23 and 24 w ith Phe as the aromatic system at the mMC1,35R. D-Napthylalanine (2’) SHU9119 is a cyclic melanocortin antagoni st developed during SAR studies of MTII.117,118,255 The only difference between the two lig ands is that SHU9119 contains a bulky naphthalene aromatic group in place of the DPhe in MTII.117,118,255 It has been suggested that the antagonist activity of SHU9119 is due to an interaction between DNal(2’) and the adjacent Arg in which the aromatic modifies the conforma tion of Arg resulting in unique molecular interactions with the mMC3R and mMC4R.43 As is depicted in Figure 5-6, the aromatic character of DPhe and DNal(2’) ar e different and interact differently with Arg and the receptors. To test the theory that a cationinteraction is invol ved in SHU9119’s antagonist activity, the Arg was substituted with Lys and Ala (peptides 25 and 26) to investigate the effect of cation strength on antagonist activity. When a Lys was substituted for Arg in com pound 25, there was a decrease in potency at the mMC1R and mMC5R as compared to Arg, but the ligand retained its agonist activity. Like

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147 SHU9119, peptide 25 is an antagon ist at the mMC3R and mMC4R, except the affinity for the receptors is decreased as eviden ced by Ki values (Ki = -log pA2).230 When Arg is replaced with Ala in peptide 26, the cationeffect is removed, and the poten cy is decreased at the mMC1, 3 and 4R, though antagonist activity was retained at the mMC3R and mMC4R. At the mMC5R, however, there was no significant difference betw een peptides 17 and 26 indicating that the cationinteraction may not be as important for activ ity at this receptor. Figure 5-9 shows a bar graph representation of these data. Figure 5-9. Bar graph of peptides 17, 25 and 26 w ith DNal(2’) as the aromatic system at the mMC1,3-5R. Values at the mMC3R and mMC4R are antagonist Ki values. Napthylalanine (2’) Peptides 18, 27 and 28 differ from the DNal(2’) compounds only in the positioning of the aromatic moiety. In this series an LNal(2’) was used in conj unction with Arg, Lys, and Ala. Interestingly, while the D conformation of the aromatic produces peptides which are potent antagonists at the mMC3R and mMC4R, changing the conformation to the L form results in compounds with significantly decreased agonist activity. The expected Arg > Lys > Ala potency series was observed at all receptors, though the Al a peptide 28 was a high micromolar agonist at the mMC3R and did not possess any stimulatory activity at the mMC4R. The most potent compound in this series is the Arg peptide 18 at the mMC5R, with low nanomolar activity.

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148 Substitution of the Arg resulted in significant de creases in potency, however. This is different from the other mMC5R series observed so far because the cationinteraction seems to be more important when the aromatic is Nal(2’) than wh en it is DPhe, Phe, or DNal(2’). A bar graph representation of these da ta is found in Figure 5-10. Figure 5-10. Bar graph of peptides 18, 27 and 28 with Nal(2’) as the aromatic system at the mMC1,3-5R. D-Napthylalanine (1’) The next aromatic system investigated was DNal(1’) (peptides 19, 29 and 30). DNal(1’) differs from DNal(2’) in the point of attachment of the napthyl ring to th e methyl linker. This results in a different interaction with the Arg cation and with the receptor. At the mMC1R, all three peptides were either subor low-nanomol ar agonists, with no difference between the Arg and Lys peptides and only a slight 8-fold decreas e in potency for the Ala peptide. At the other three receptors, all peptides were agonists and th e potency series Arg > Lys > Ala is maintained, indicating the importa nce of the cationinteraction at these receptors.

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149 Figure 5-11. Bar graph of peptides 19, 29 and 30 with DNal(1’) as the aromatic system at the mMC1,3-5R. Napthylalanine (1’) The final aromatic amino acid that was in cluded was Nal(1’) (p eptides 20, 31 and 32). When compared with the D conformation of th is amino acid, the poten cy was significantly decreased with the L form. Additi onally, at the mMC1R there is li ttle change in potency between Arg, Lys, and Ala. At the mMC3R, the Arg compound is the most potent, though the Lys and Ala compounds are approximately equipotent. An in teresting effect is seen at the mMC4R. The expected Arg > Lys agonist trend was seen with the first two peptides, however the Ala peptide 32 was a partial agonist and antagonist at the mM C4R. There may be other unique interactions that are taking place between Nal(1’), Ala and the receptor which accounts for this antagonist activity. At the mMC5R the expected Arg > Lys > Ala trend is maintained.

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150 Figure 5-12. Bar graph of peptides 20, 31 and 32 with Nal(1’) as the aromatic system at the mMC1,3-5R. The Ala compound 32 at the mMC4 R is expressed as an antagonist Ki value. Conformation Effects An important observation that should be made from this study is that for each aromatic system investigated, the D form of the amino aci d is significantly more potent than the L form. This is in accordance with previous studies investigating conformational differences at the aromatic position, especially in the case of the development of NDP-MSH from -MSH.303 Holder et al. reported the differences between D and L conformations of the napthylalanine derivatives (see Table 5-1), and also recognized the significant increase in potency when the L form was replaced with the D form.7 It is apparent that the D fo rm of the aromatic amino acid allows for a more favorable interaction with the melanocortin receptors resulting in greater stimulatory activity. Receptor Selectivity Each receptor is unique in the way it responds to a particular ligand. Each melanocortin receptor has been characterized based on how it interacts with each endogenous and synthetic ligand and this is often the basis for the specific physiolo gical activity of each receptor. It should be expected, therefore, that the st rength and importance of the cationinteraction at each receptor should be unique.

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151 At the mMC1R, four aromatics: DPhe, Phe, DNal(2’) and Nal(2’) exhibited the hypothesized Arg > Lys > Ala potency series which suggests the importance of the cationinteraction for activity. However, when the ar omatics DNal(1’) and Nal(1’) were used, the cationeffect was greatly decreased. In the case of DNal(1’) the peptides still maintained subnanomolar activity, indicating that there are other important interac tions responsible for activity at this receptor. Cationinteractions at the mMC3R seem to be very important for both agonist and antagonist activity. The only aromatic system th at does not follow the Arg > Lys > Ala potency series is Nal(1’). Like the mMC3R, the Nal(1’ ) system appears to interact through a unique mechanism. The only aromatic which exhibited antagonist activity was DNal(2’). No other napthyl derivatives had any antagon ist activity. It is concluded, therefore, that DNal(2’) is unique in the way it interacts with Arg and the receptor to act as an antagonist. The mMC4R is the receptor at which cationinteractions seem to play the most important role in agonist activity. For all aromatic syst ems tested, Arg > Lys > Ala for both agonist and antagonist activity. Additionally, DNal(2’) was the only aromatic system to have antagonist activity for all three peptides. No other napthyl derivatives had anta gonist activity at this receptor except for peptide 32, which contains the Nal( 1’)/Ala combination. It is unknown why this peptide has antagonist activity, but may be a re sult of the local unique environment created by this peptide. The mMC5R has mixed cationspecificities. When the aromatic is DPhe, Phe, or DNal(2’) the cationinteraction does not appear to be a significant factor affecting potency. The other aromatics Nal(2’), DNal(1’), and Nal(1’), however, appear to interact through a cation

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152 interaction at this receptor. It is suspected that this is due to uni que ligand-receptor interactions at the mMC5R. Agonist vs. Antagonist Selectivity Part of the original hypothesis for this study i nvolved the agonist vs. antagonist selectivity between MTII and SHU9119.117,118,255 It has been proposed that the antagonist activity of SHU9119 is due to a unique interaction betw een the bulky aromatic DNal(2’) group and the cationic character of Arg.43 Based on the results of this st udy, it was observed that DNal(2’) is the only napthyl derivative in this work that had significant antagonist activity that was significantly affected by a cationinteraction. The aromatic systems Nal(2’), DNal(1’) and Nal(1’) were not antagonists ex cept for peptide 32, which was a weak antagonist at the mMC4R. Conclusions Several conclusions may be drawn from the information obtained in this study. First it appears that the original hypothesis is corre ct and there may be a significant cationinteraction between the Arg and the adjacent aromatic am ino acid in melanocortin ligands. This was confirmed by the observation that in the majority of peptides synthesized in this study the Arg compound was the most potent, followed by Lys, and finally Ala as the least potent compound. It was also observed that in each aromatic system tested, the D form of the amino acid was more potent than the L form. Additionally, DNal(2’) is the only aromatic system tested which has antagonist activity at the mM C3R and mMC4R. An interest ing observation is that the Nal(1’)/Ala combination results in a peptide with antagonist activity at the mMC4R. These studies are significant as they pr ovide important information which may be used in the future to design new peptide and non-peptide liga nds for the melanocortin receptors.

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153 CHAPTER 6 DESIGN OF PEPTIDOMIMETICS AND SM ALL MOLECULES: 1,4-BENZODIAZEPINE2,5-DIONES AS nM MELANOCORTIN AGONISTS Portions of this chapter are “in press” in the Journal of Medicinal Chemistry 1,4Benzodiazepine-2,5-diones were sy nthesized, purified, and analytic ally characterized by Krista R. Wilson in the Haskell-Luevano laborato ry. Pharmacology was performed by Federico Portillo, Michael S. Wood, Ni cholas B. Sorenson, Dong V. Phan, and Zhimin Xiang of the Haskell-Luevano laboratory. NMR analysis was done by Krista R. Wilson, Christine G. Joseph, and Rachel M. Witek of the Haskell-Luev ano laboratory. NMR of compounds 40, 46, 47, 52, and 56 were performed by Jim Rocca of the Univ ersity of Florida NMR Core Facility. Data analysis was performed by Krista R. Wils on and partially by Carrie Haskell-Luevano. Introduction Figure 6-1. Structures of A. 1,4-benzodiazepine2-one, B. 1,5-benzodiazepine-2-one and C. 1,4benzodiazepine-2,5-dione templates. The 1,4-benzodiazepines are an import ant class of privileged templates,35 and numerous derivatives have been identified th at have selective activities agai nst a diverse array of biological targets.36-40 A subset of the 1,4-benzodiazepines, the 1,4-benzodiazepine-2,5-diones (Figure 61C), are the focus of this work. 1,4-Benzodiaz epine-2,5-diones have been reported to possess anticonvulsant, anxiolytic, and antitumor propertie s, as well as being ch olecystokinin receptor (CCK), opiate receptor and platelet glycoprotein IIb-IIIa antagonists,190-193 as well as having herbicidal properties.194 In addition, the 1,4-benzodiazepine-2,5-dione core appears in a number of natural products.195-197

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154 Information obtained from peptide related SAR studies may be used in the design of peptidomimetic and small molecule templates that are potent and selec tive for the melanocortin receptors. Peptides are not ideally suited for use as drugs due to their large molecular weight, high rate of first-pass metabolism in the stomach and small intestine, and extreme sensitivity to changes in pH or temperature; therefore it is desirable to obtain a small molecule drug. In the design of small molecules, the information obtain ed from the peptide SAR studies is used to create conformationally restrained peptides and/ or small molecules. Further studies are done to optimize the molecule in regards to potency, sele ctivity, and toxicity. Small-molecule libraries have become an important part of the drug discovery process190,304-306 with particular emphasis placed upon the preparation and evaluation of libraries based upon privileged structures.35 These structures display a number of different functio nalities that result in a number of potent and specific drugs or candidates for different therapeutic targets.35,190,307 Structure-activity relationship studies of the endogenous melanocortin agonists have shown that these ligands all contain the core tetrapeptide His-Phe-Arg-Trp.114,115 Based on these studies, synthetic peptide agonists of the melanoc ortin receptors were designed which are even more potent than the endogenous peptides, and contain a DPhe in place of the endogenous Phe.4 NMR studies of both endogenous and synthetic liga nds have indicated the presence of a reverse turn mimetic in the Phe-Arg-Trp region.308-310 A reverse turn, or -turn, is a secondary structural motif found in peptides and proteins, and invol ving four amino acid side chains, termed i i +1, i +2 and i +3.18 The carboxyl carbon of i and the N-H of i +3 are often hydrogen bonded to increase the stability of the structure. The re verse turn is defined by the following conditions: positions i i +1, i +2, and i +3 are not in an -helix, and the distance between C ( i ) and C ( i +3) is less than 7.18

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155 Figure 6-2. Small molecules with activity at the melanocortin recep tors and based on privileged structure templates. Compound References: A,26 B,26 C,27 D,311 E,312 F,313 G,314 H,315 I,316 J,317 K,318 L,319 M,320,321 N,320,321 O,322 P,323 Q.324 The presence of -turns in melanocortin peptides has been well documented. NMR modeling studies have predicted a -turn structure to be present in the His-Phe-Arg-Trp region of -MSH,308-310 NDP-MSH,325 MTII,325 and in other melanocortin analogues. Additionally, nonpeptide libraries have been constructed which incorporate -turns that are melanocortin

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156 agonists.26,27 The first heterocyclic nonpeptide molecules that were identified as melanocortin agonists were based on a 9-membered hete rocyclic ring system (Figure 6-2A,B).26 These compounds were based on a -turn motif, and were shown to be micromolar agonists at the mMC1R.26 Bondebjerg et al. also did a study in which a thioethe r cyclized scaffold was used to mimic the -turn found in the endogenous and sy nthetic peptides (Figure 6-2C).27 These molecules were shown to be nanomolar agonists at the mMC1R, and the mMC3-5R.27 These results indicate the importance of the reverse turn in the design of melanocortin ligands. As there are only a few bioactive conformatio ns able to stimulate the recepto rs, it is desirable to design ligands with a more rigid template, which may in crease the concentration of bioactive molecules in solution. The 1,4-benzodiazepine-2,5-dione template used in this study has been shown both to exhibit a reverse turn and to adopt a more rigid, planar conformation.326,327 The scaffold adopts a reverse turn-like conformation, enabling th e modification of side chains to mimic the 3dimensional conformation of amino acid side chai ns found in bioactive peptides. In this study, the template was modified to mimic the DPhe-Arg-Trp tetrapeptide in synthetic melanocortin ligands. The term “privileged structure” was first coined in 1988 by John Ellman as a way to describe molecular substructures that were able to provide a framework for potent and selective ligands at multiple receptors.35 At the time Ellman was studying the benzodiazepine structure, trying to develop potent CCK antagonists.35 He noticed that there were subtle differences between structures that were active at the CCK, gastrin, or cen tral benzodiazepine receptors. NMR analysis of the 3-D conformations of thes e molecules led him to hypothesize that these “privileged” substructures could be functionali zed to create ligands that make specific interactions with different receptors.35 Since then, extensive library analyses have identified a

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157 plethora of substructures commonly found in sm all molecule drugs (See Figure 1-14 for more privileged templates). Small molecule ligands based on many different privileged templates are shown in Figure 6-2. Figure 6-2D depicts an MC 4R agonist containing the 2-phe nyl-indole privileged template.311 Another template, the spiro-pipe ridine-indane substructure, was used in the design of Figure 62E, which is an agonist at the MC4R.312 Piperidine and its derivatives have been used to design several melanocortin ligands. Dyck et al. synthesized a series of aryl piperidine derivatives based on growth hormone secretagogue compounds that were potent and selective for the MC4R (Figure 6-2F).313 Another group designed melanocortin liga nds from the bis-piperazine template that were meant to inhibit th e binding of AGRP to the MC4R. Though this end was not realized, the compound in Figure 6-2G was reported to antagonize the MC4R better than AGRP.314 Richardson et al. also used the piperazine template to design potent MC4R ligands (Figure 62H).328 Finally, Xi et al. used a series of piperazine-succi namide derivatives to create small molecules with nM activity at the MC4R (Figure 6-2I).329 Merck scientists developed a gr oup of molecules that contained the piperidine core as well as a tetrazole ring.317 The compound in Figure 6-2J was ab le to selectivel y agonize the MC4R over the other melanocortin subtypes.317 Herpin et al. was able to design small molecules based on piperidine that were selective for the MC1R over the MC4R (Figure 6-2K).318 Finally, Ujjainwalla et al. reported a group of compounds that co ntained the pyridazinone core and was able to stimulate the MC4R.319 Optimization of these com pounds by Merck resulted in compounds with improved binding and pot ency at the MC4R (Figure 6-2L).319 Several other templates have been used with some success. The tetrahydropyran scaffold was used to create the compound in Figure 6-2M which is a partial agonist at the MC4R,320,321

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158 as well as the compound in Figure 6-2N, which mi mics the side chain of arginine, but was only able to stimulate the MC1R at hi gh concentrations and not the MC4R.320,321 Pan et al. designed compounds containing a 1,2,4-thiazolium ring which wa s able to bind MC4R and when injected into rats resulted in a decreas e in food intake (Figure 6-2O).322 Vos et al. used a cyclocondensed imidazole ring to design antagonists of the MC4R which were orally active and concentrated in the brain (Figure 6-2P).323 Finally, Cain et al. has recently reported a peptide mimetic that mimics the -turn secondary structure and was able to selectively stimulate the MC1R (Figure 62Q).324 It is clear that there is a str ong precedent for the use of privil eged structures in the design of melanocortin ligands. Theref ore it was hypothesized that a library of small molecule 1,4benzodiazepine-2,5-diones may be created that mimic the side ch ains of the His-Phe-Arg-Trp melanocortin agonist core tetrapeptide resulting in agonist activity at the melanocortin receptors. Christine G. Joseph, a former graduate student in the Haskell-Luevano lab or iginally investigated this template as a way to create selective melanocortin ligands (Table 6-1). In order to achieve these goals, a number of synthetic schemes for the solid-phase organic synthesis of benzodiazepines were evaluated.191-194,198-203,330 The optimized synthesis approach utilized is shown in Schemes 6-1 and 6-2.190,194,200,304 It begins with the attachment of the -amino ester to the solid support, followed by acylation with an an thranilic acid, base-catalyzed lactamization, alkylation, and cleavage. The sequence relies on th e incorporation of three different components, anthranilic acids (R1), al kylating agents (R2), and -amino esters (R3) which are available commercially with appropriate side-chain protec tion. Synthesis with this scheme also provided high yield and relatively clean cr ude products. Several compounds we re synthesized, analytically characterized and pharmacologically characterized by Christine G. Joseph for functional agonist

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159 activity at the melanocortin receptors (MC1R, an d MC3-5Rs). The results are shown in Table 61. Scheme 6-1. Solid-phase organic synt hesis of 1,4-benzodiazepine-2,5-diones. Scheme 6-2. Synthesis of acetanilide-lithium salt. Based on the results of Dr. Joseph’s work in Table 6-1, an additional twenty compounds were designed by Krista Wilson to investigate the structural requirements necessary for activity at the melanocortin receptors, these are shown in Figure 6-3. Three aminobenzoic acid derivatives were used at the R1 position: a hydrogen, a 9-methyl, and an 8-chloro; five different alkylating agents were used at the R2 position: benzyl, biphenyl napthyl, propyl, and butyl; and three amino acid esters were used at the R3 position: benzyl (phenylalanine), propyl guanidine

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160 (arginine), and 4-aminobutyl (lysine) (Figure 6-4). It was hypothesize d that these compounds would allow the exploration of 3-D space in th e molecular recognition and activation of the melanocortin receptors by the 1,4-benzodiazepine-2,5dione class of privilege d structures and to determine structural requirem ents for this interaction.

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161 Figure 6-3. Structures of 1,4-benzodiazepine-2,5 -diones synthesized in this study by KRW.

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162 Figure 6-4. Structures of building blocks us ed to synthesize 1,4-benzodiazepine-2,5-diones 3958.

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163Table 6-1. Pharmacology data of compounds synthesized by Dr. Christine Joseph. Agonist EC50 (M) Compound Structure mMC1R mMC3R mMC4R mMC5R -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe -Arg-Trp-Gly-Lys-Pro-Val-NH2 0.00070.00023 0.002140.00046 0.002560.00029 0.00200.00020 Template: R1 R2 R3 mMC1R mMC3R mMC4R mMC5R 33 H Benzyl Benzyl 32.98.2 No agonist No agonist No agonist 34 H Benzyl Propyl guanidine 16.56.0 No agonist No agonist No agonist 35 H Benzyl 1H-indol-3-ylmethyl 4.62.5 No agonist No agonist No agonist 36 9-Methyl Benzyl 1-Benzyl-1Hindol-3-yl-methyl 21.66.9 No agonist No agonist No agonist 37 8-Chloro Benzyl 4-aminobutyl 0.0480.023 0.320.08 0.240.075 0.0870.038 38 8-Chloro Napthalene-2yl-methyl 1H-indol-3-ylmethyl No agonist No agonist No agonist No agonist Indicated errors represent the standard error of the mean, determ ined from at least four indepe ndent experiments. “No agonist” indicates that no agonist activity was observed at up to 100 M. Agonists that possessed some stimulatory response at 100 M concentrations are indicated as percent response relative to the maximal response ob served for the forskolin control.

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164Table 6-2. Analytical data of 1,4-benzodiazep ine-2,5-diones synthesized in this study. Compound HPLC k’ (system 1)90 HPLC k’ (system 2)90 % purity m/z (M, calcd) m/z (M+1, expt) 39 5.5 6.8 >97 336.9 336.9 40 10.9 15.3 >98 432.5 433.1 41 8.1 10.8 >98 442.5 440.6 42 7.1 10.1 >97 413.5 412.6 43 10.8 13.4 >98 406.5 405.6 44 7.1 9.2 >98 416.5 414.6 45 6.9 9.1 >98 387.5 386.7 46 7.8 10.7 >98 308.4 309.5 47 8.5 12.3 >99 322.4 323.1 48 12.1 11.4 >95 370.4 369.9 49 5.8 8.1 >97 380.4 378.7 50 5.6 7.4 >95 351.4 350.8 51 10.6 13.3 >97 390.9 389.7 52 6.6 9.5 >99 399.9 401.1 53 12.5 13.6 >98 467.0 465.7 54 9.2 12.0 >97 477.0 474.6 55 8.1 11.4 >96 448.0 446.7 56 11.3 17.2 >99 440.9 440.6 57 7.7 10.8 >95 451.0 448.6 58 8.0 12.1 >97 421.9 422.5 HPLC k’ = c[(peptide rete ntion time – solvent retention time)/( solvent retention time)] in solvent system 1 (10 % acetonitrile in 0.1 % trifluoroacetic acid/water and a gradient to 90 % acetonitrile over 35 min) or solvent system 2 (10 % methanol in 0.1 % trifluoroacetic acid/water and a gradient to 90 % methanol over 35 min). An analytical Vydac C18 column (Vydac 218TP104) was used with a flow rate of 1.5 ml/min. The peptide purity wa s determined by HPLC at a wavelength of 214 nm by comparison of the area under the curve of co mpound peak compared to all other peaks. Mass spectroscopy was performed on a Voyager-DE Pro (University of Florida Protein Core).

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165Table 6-3. Pharmacology data of 1,4-benzodiazepine-2,5diones synthesized in this study by Krista Wilson. Agonist EC50 (M) Compound Structure mMC1R mMC3R mMC4R mMC5R -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe -Arg-Trp-Gly-Lys-Pro-Val-NH2 0.00070.00023 0.002140.00046 0.002560.00029 0.00200.00020 Template: R1 R2 R3 mMC1R mMC3R mMC4R mMC5R 39 H Benzyl 4-aminobutyl 50%@100M No Agonist No Agonist No Agonist 40 H Biphenyl-2-yl-methyl Benzyl 0. 230.0027 4.20.07 1.971.23 60%@100M 41 H Biphenyl-2-yl-methyl Propyl gua nidine 38.88.9, 70% No Agonist No Agonist No Agonist 42 H Biphenyl-2-yl-methyl 4-a minobutyl 59.515.9 No Agonist No Agonist 60%@100M 43 H Naphthalen-2-yl-methyl Benzyl 50%@100M No Agonist No Agonist 60%@100M 44 H Naphthalen-2-yl-methyl Propyl guanidine 34.210.1, 70% No Agonist No Agonist 70%@100 M 45 H Naphthalen-2-yl-methyl 4-a minobutyl 71.620.1 No Agonist No Agonist 69.718.3, 75% 46 H Propyl Benzyl 6.92.6 80%@100M 3516 3.31.0 47 H Butyl Benzyl 70%@100M No Agonist No Agonist No Agonist 48 9-Methyl Benzyl Benzyl 50%@100M No Agonist No Agonist No Agonist 49 9-Methyl Benzyl Propyl gua nidine 60%@100M No Agonist No Agonist No Agonist 50 9-Methyl Benzyl 4-aminobutyl 0.006.004, 50% No Agonist No Agonist No Agonist 51 8-Chloro Benzyl Benzyl 36.34.9, 75% No Agonist No Agonist 60%@100M 52 8-Chloro Benzyl Propyl guanidine 1.80.10 30.723 60%@100M 174.5 53 8-Chloro Biphenyl-2-yl-methyl Benzyl 50%@100M No Agonist No Agonist No Agonist 54 8-Chloro Biphenyl-2-yl-methyl Pr opyl guanidine 20.54.7, 75% No Agonist 50%@100M No Agonist 55 8-Chloro Biphenyl-2-yl-methyl l 4-aminobutyl No Agonist** No Agonist No Agonist No Agonist 56 8-Chloro Naphthalen-2-yl-methyl Benz yl 4.71.3 23.32.5 80%@100M 4.351.7 57 8-Chloro Naphthalen-2-yl-methyl Propyl guanidine 60%@100M No Agonist No Agonist 70%@100M 58 8-Chloro Naphthalen-2-yl-methyl 4-ami nobutyl 3.40.07 30%@100M 30%@100M 65%@10M Indicated errors represent the standard error of the mean, deter mined from at least four independent experiments. “No agonist” indicates that no agonist activity was observed at up to 100M. Agonists that possessed some stimulatory response at 100 M concentrations are indicated as percent response relative to the maximal response observed for the forskolin control. **Compound 59 was toxic at the mMC1R at 100M.

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166 Results Analytical characterization and agonist functional data for the 1,4-benzodiazepine-2,5dione molecules synthesized in this study are summarized in Ta bles 6-2 and 6-3, respectively. All functional agonist data are expressed as M EC50 values with the associated standard error of the mean (SEM) derived from at least three independent experiments provided. 1H-NMR and proton assignments of compounds 3958 may be found in Appendix A. Compound 39 contains a hydrogen at R1, a benzyl group at R2, and a 4-aminobutyl group at R3. At the mMC1R, compound 39 exhibits 50 % maximal response. No stimulatory response was observed at the mMC3-4R, but some agonist activity was seen at the mMC5R, with 40% maximal response as compared to the forskolin control at up to 100 M concentrations. Compounds 40-42 contain a hydrogen at the R1 position, a biphenyl-2-yl-methyl group at the R2 position and are differentially modifi ed at the R3 position. Compound 40, containing a benzyl moiety at the R3 position, is a nM agoni st at the mMC1R, is 18-fold mMC1R versus mMC3R selective, is ca 9-fold mMC1R versus mMC4R selective, exhibits M agonist potency at the mMC3R and the mMC4R, and is able to stimulate a 60% maximal forskolin response at the mMC5R. When the R3 position is substitu ted with a propyl guanidine group in compound 41, the molecule now is a M agonist at the mM C1R with 70% maximal activity, and exhibits no activity at the mMC3-5R. When R3 is a 4-am inobutyl group in compound 42, the molecule is a full micromolar agonist at the mMC1R, and posse sses some stimulatory activity at the mMC5R, but no activity at the mMC3-4R. Compounds 43-45 have an H in the R1 position, and a naphthalene-2-yl-methyl moiety at the R2 position, but they differ at R3. When R3 is a benzyl in compound 43, there is no stimulatory activity at the mMC3-4R, but 50% and 60% maximal activity was observed at the mMC1R and mMC5R respectively compared to maximal forskolin response. Compound 44

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167 contains a propyl guanidine group at position R3, a nd is a micromolar agonist with 70% maximal activity at the mMC1R. There is no activity at the mMC3-4R and only 70% maximal activity was observed at the mMC5R. Compound 45 contains a 4-aminobutyl group at R3, and is a high M agonist at the mMC1R. It does not contain any stimulatory activity at the mMC3-4R, and a micromolar agonist with 75% maximal response at 100 M as compared to forskolin control. Compounds 46-47 each have an H at R1 and a be nzyl moiety at R3, though they differ at the R2 position. When R2 is a propyl group in compound 46, M full agonist activity is observed at the mMC1R, mMC4R, and mMC5R with onl y a 80% maximal forskolin response at the mMC3R. When R2 position contains the but yl group in compound 47, only a 70% maximal forskolin stimulation response is observed at the mMC1R and no stimulatory response is observed at any of the other receptors. Compounds 48-50 each contain a 9-methyl group at the R1 position and a benzyl at the R2 position, but differ at R3. When R3 is a benzyl group in compound 48, there is only 50% maximal stimulation at the mMC1R as compared to forskolin control, and no stimulatory response observed at the mMC3-5R. When position R3 contains a propyl guanidine moiety as in compound 49, 60% maximal activity is observed at the mMC1R, though no stimulatory effect was observed at the mMC3-5R. Compound 50 contai ns a 4-aminobutyl group at the R3 position, and interestingly is a nanomolar partial agonist at the mMC1R, but only 50% maximal activity was observed. No activity was seen at the other receptors. Compounds 51-52 contain an 8-chlo ro substitution at R1 and a benzyl at R2, but differ at R3. Compound 51 contains a benzyl at R3, and is a micromolar agonist with 75% activity at the mMC1R. 51 does not stimulate the mMC3-4Rs, a nd exhibits only 60% ma ximal stimulation at the mMC5R as compared to forskolin contro l. Compound 52, containi ng a propyl guanidine

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168 group at R3, resulted in full M agonist activity at the mMC1, mMC3, and mMC5 receptors, and 60% maximal activity at the mMC4R. Compounds 53-55 all contain an 8-chloro gr oup at the R1 position and a biphenyl-2-ylmethyl moiety at the R2 position, but differ at R3. When R3 is a benzyl group in compound 53, no agonist activity was observed at the mMC3 -5R, and only weak stimulatory activity was observed at the mMC1R. Compound 54 contains a propyl guanidine group at the R3 position, and exhibits M agonist activity at the mMC1R, but no stimulat ory activity was observed at the mMC3-4R, and only slight stimulatory activit y was observed at the mMC5R. When the R3 position is substituted with a 4-aminobutyl grou p in compound 55, there is no agonist activity observed at the mMC1,3-5R. Interestingly, at the mMC1R, compound 55 was toxic at 100M concentrations. Compounds 56-58 contain an 8-chloro at th e R1 position and a napthylene-2-yl-methyl group in the R2 position, with differential subs titution at R3. Compound 56, containing a napthyl side chain at R2 and a benzyl group at R3, resulted in a M agonist at the mMC1R, mMC3R, and mMC5R but was only able to stimulate the mMC4R to 70% maximal activity observed for the forskolin control at 100 M concentrations. Compound 57 c ontains a propyl guanidine group at R3 and only exhibits weak stimulatory ac tivity at the mMC1R and mMC5R, with no activity at the mMC3-4R. Compound 58, differing from compound 56 by the presence of the 4-amino butyl moiety at the R3 position, is equipoten t with compound 56 at the mMC1R yet was only able to generate weak stimulatory respons es at the mMC3R, MC4R, and mMC5R at 100 M concentrations. Discussion The benzodiazepine template has been widely studied as a scaffold for non-peptide drugs.35 The core was first identified as a CCK anta gonist with the isolation of asperlicin, a

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169 natural product from Aspergillus alliaceus .331-333 Though it showed antag onist activity at the CCK receptor, asperlicin did not exhibit good bioa vailability, leading researchers to develop new ligands based on the benzodiazepine core. Many of these compounds have been shown to exhibit better pharmacokinetic properties than the natura l product. Additional studies have resulted in compounds that are selective for the CCKA (gut ) receptor versus the CCKB (brain) receptor.334339 Derivatives have been identified wh ich have properties as anxiolytics, -opioid receptor agonists,41 oxytocin recept or antagonists,37,38 endothelin antagonists,340 HIV Tat antagonists,39 and reverse transcriptase inhibitors40 among others. As many targ ets are members of the Gprotein coupled receptor super fa mily (i.e. cholecystokinin, -opioid, oxytocin, and endothelin receptors), it was hypothesized that benzodi azepine derivatives could be designed as melanocortin ligands. Traditionally, benzodiazepines have been used as anxiolytics and sedatives, in which they serve as allosteric enhancers of the GABA recep tors. In this role, benzodiazepines bind to a distinct site on the GABA receptor, wh ere they potentiate the effect of -aminobutyric acid (GABA), allowing for increased opening of pos tsynaptic chloride channels leading to hyperpolarization of cell membranes and resul ting in sedative and hypnotic CNS effects. The lipophilic nature of these compounds combined with their small si ze enables them to cross the blood brain barrier (BBB), and exer t their CNS effects. As the hMC4R is centrally expressed, a small molecule melanocortin drug would need to cross the BBB in order to bind to their target receptors. As the benzodiazepine te mplate is able to cross the BBB and has been shown to have activity at other GPCRs, it may be possible to de sign small molecule ligands of the melanocortin receptors with significant in vivo CNS activity. One caveat of this template may be presence of undesirable side effects due to binding to the GABA receptor.

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170 The 1,4-benzodiazepine-2,5-dione library synt hesized in this study was designed to interact with proposed binding sites in th e melanocortin receptors. Homology molecular modeling of the mMC1R44,341 and the mMC4R43,48-50,342 suggest the presence of hydrophobic and electrostatic binding pockets whic h are essential for binding of the melanocortin ligands to the receptor. Based on the results of this study, it is proposed that the p(Cl)-phenyl ring of the benzodiazepine and an aryl side chain may in teract with the hydrophobi c pockets and that a cationic component may interact with the anionic binding pocket. The benzodiazepines reported herein were de signed based on a set of 1,4-benzodiazepine2,5-dione compounds synthesized by Christine G. Joseph of the Haskell-Luevano laboratory that were shown by functional analysis to have activ ity at the melanocortin receptors. These results are reported in Table 61 and a summary follows. Compounds 33-38 in Table 6-1 represent thos e compounds synthesized by Christine G. Joseph. These studies revealed four compounds selective for the mMC1R and one compound with nanomolar activity at all melanocortin receptors. Co mpounds 33-35 are identical in that they all contain a hydrogen at position R1 and a benzyl at R2. They differ only at the R3 position, where 33 contains a benzyl, 34 a pro pyl guanidine, and 35 a 1H-indol-3-yl-methyl. These compounds were micromolar agonists at the mMC1R and had no activity at any of the other receptors. Potency increased from 33 thr ough 35, showing the potency series 1H-indol-3yl-methyl > propyl guanidine > benzyl where the indole co mpound is the most potent. Additionally, R1 substituents were varied, incorporating either a H, a 9-methyl, or an 8-chloro group. Compounds 33 and 36 were identical except fo r the substitution of a 9-methyl group for the hydrogen in compound 36. This compound was le ss potent than 33, but still maintained micromolar agonist potency at the mMC1R. When R1 was changed to an 8-chloro in compound

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171 37, there was nanomolar activity observed at all melanocortin re ceptors tested, making this compound very promising for lead optimization. Fi nally, when a naphthalene group was inserted into position R3 in compound 38 there was no activity observed at a ny of the receptors. Figure 6-5. Structure of Compound 37, a nanomolar agonist at all melanocortin receptors tested. These studies are highly signi ficant as they have identifie d a small molecule compound, 37, which possesses nanomolar activity at the mMC1,3-5R (Figure 6-5) Micromolar mMC1R agonists were also identified. Based on these in teresting pharmacology results, a new series of compounds (Compounds 39-58) were designed in order to investigate the st ructural requirements necessary for benzodiazepine activity at the mela nocortin receptors, and to probe the structureactivity relationships at each point of diversity. The numbering system used as well as the location for the three points of di versity are shown in the figure labeled “Template” in Table 6-3. Building blocks are shown in Figure 6-4. Position R1. Three different structural elements were used at position R1: a hydrogen (compounds 39-47), a 9-methyl (compounds 48-50), and an 8-chloro (compounds 51-58). This position was derived from the substituted 2-ami nobenzoic acids used during synthesis (Figure 64). Electronically, these substitu ents represent three different electronic effects when coupled with an aromatic system. The hydrogen does not cont ribute any electronic effects to the aromatic ring, so the contribution to the pharmacophore repr esents only an aromatic ring. The methyl group, however, is an electron-dona ting substituent, which may lead to an increased electron

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172 density on the face of the aromatic system. The 8-chloro represents a highly electronegative moiety, which is electron-withdrawing, thereby decreasing the electron density on the aromatic face. Electronegative halogens have been shown to act as electron acceptors, and may interact with the bonds of aromatics or with the systems of peptide bonds. They may also interact with the lone pairs of N, O, or S. Studies of stacking in peptides have suggested that the addition of an electron-withdrawing group in the para position on a phenyl ring increases activity.343-345 Addition of chlorine creates an elec tron-deficient ring which may interact favorably with the relatively el ectron-rich ring of tyrosine.346 Based on this information, it may be proposed that the halogenated ring of the benzodiazepine template may interact with the receptor to create a favorable -stacking charge-transfer relati onship, resulting in increased agonist activity. When introduced into the 1,4-benzodiazepine2,5-dione template, some general trends were observed. First is that when R1 is a hydrog en, almost all compounds have some activity at the mMC1R and mMC5R, but no activity at th e mMC3-4R. Exceptions are compounds 40 and 46, which exhibit activity at all melanocortin re ceptors tested. Compounds substituted with a 9methyl at R1 followed a similar trend, with all three compounds tested having activity only at the mMC1R. When R1 was an 8-chloro, the pharmacology was more complex, with some compounds having activity at betw een one and four receptors, and other compounds with no activity observed. Due to the complex pharm acology observed, it is proposed that though position R1 may be important for activity in combin ation with other factors, it does not appear that position R1 alone is respons ible for individual effects, though the 8-chloro group does seem to confer more activity at the mMC3-4R, while the hydrogen and 9-methyl have more activity at the mMC1R, and some at the mMC5R.

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173 Position R2. Another level of diversity was added at position R2. These five groups were derived from alkylation with the corresponding al kyl or aryl halide (Figure 6-4). The bromide derivatives were used for the benzyl, biphenyl, and na pthyl moieties, and th e iodo derivative was used to add the propyl and butyl groups due to low reactivity of the brom o derivatives. Each of these derivatives was chosen for a reason based on previous peptide SAR data. The benzyl group was chosen due to its similarity with the Phe in the core tetrapeptide sequence His-Phe-Arg-Trp, which has been shown many times to be essent ial for activity at the melanocortin receptors.116 The biphenyl group, when substituted for Phe in the tetrapeptide Ac -His-Phe-Arg-Trp-NH2 has been shown to stimulate the mMC1, 4, and 5 rece ptors with nanomolar activity when in the D form, and to have micromolar activity at the mMC1,5R in the L form.7 Napthyl derivatives have been shown to have significant effects on pha rmacology when introduced into melanocortin peptides. When DNal(2’) was introduced into th e synthetic cyclic agonist MTII, the molecule retained agonist activity at the mMC1,5R, but was converted into a potent antagonist at the mMC3,4R.116-118,255 Studies of the melanocortin tetrapeptide7 and other melanocortin ligands142,347 have identified more templates that are converted into antag onists at the mMC3,4R by addition of a DNal(2’). Interes tingly, use of the L form of Na l(2’) or the Nal(1’) derivative results in ligands which are agonists at the melanocortin receptors, but with a significant decrease in potency as compared to Phe or DPhe derivatives.7 Propyl and butyl derivatives were chosen to see the effects of an alkyl substituent. Aliphatic chains have been shown to increase the potency and prolong the effects of the tetrapeptide His-Phe-Arg-Trp-NH2 in vitro .34 Additionally, it was desired to see the difference of an alkyl and aryl group at this position. From the compounds synthesized by Christine G. Joseph, it appeared that the combination of hydrogen at R1 and a benzyl at R2 resulted in compounds with activity at the mMC1R (see

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174 compounds 33-38, Table 6-1). This assessment seems to be accurate since compound 39 also had activity at the mMC1R and at no other receptors. When a benzyl at R2 was combined with a 9methyl at R1, Dr. Joseph re ported compound 36, which also ha d activity at only the mMC1R. Compounds 48-50 also contained this combina tion and were agonist s at the mMC1R only. Finally, when Dr. Joseph coupled a benzyl with an 8-chloro at R1, compound 37 was created, which possessed nanomolar activity at all melanocortin receptors. In response, compounds 51 and 52 were synthesized. Compound 51 exhibited some activity at the mMC1,5R and no activity at the mMC3,4R, and compound 52 had micromolar activity at the mMC1, 3, and 5R, but only slight stimulatory activity at the mMC4R. From these studies it may be inferred that combining a benzyl at position R2 with either a hydrogen or 9-methyl at R1 result s in compounds which are only active at the mMC1R. Compounds with an 8-chlo ro at R1 and a benzyl at R2 were shown to have increased activity at the mMC3,4R over th e other compounds and appears to be important for conferring activity at the mMC3,4R. The next R2 group that was investigated wa s the biphenyl moiety. When coupled with a hydrogen at R1, three compounds with distinct pharmacology were obtained (compounds 40-42). These compounds differed significantly in their activity at each receptor, and thus it is concluded that the differences are due to the R3 position and will be discussed below. When the biphenyl was grouped with an 8-chloro at R1, com pounds 53-55 were obtained. These compounds had less activity than the hydrogen/ biphenyl compounds and only two had activity at the mMC1R. Compound 55 had no activity at any of the receptors tested and in fact was toxic at the mMC1R, causing cells to die during stimul ation. The 8-chloro/biphenyl comb ination does not appear to be tolerated well, and effects are highly variable based on the R3 substituent.

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175 Only one compound containing a napthyl group at the R2 position was synthesized by Dr. Joseph (compound 38) and this compound did not have any activity at the melanocortin receptors tested. Since the napthyl moiety has been shown to convert ligands into melanocortin antagonists,117,118,255 this compound was also tested for antagonist activity at the mMC3,4R, however no activity was observed. To further in vestigate the effects of this group on ligand potency, the napthyl was combined with either a hydrogen or 8-chloro at the R1 position. Compounds 43-45 contain the hydrogen/napthyl combination and all compounds were micromolar mMC1R and mMC5R agonists. Th ese results were very similar to the hydrogen/biphenyl combination and it does not app ear that one is preferred over the other, though the benzyl compounds were more poten t than either. Compounds 56-58 possess an 8chloro at R1 and a napthyl at R2. Although compound 57 only weakly stimulates the mMC1,5R, there is no activity at the mMC3,4R. Compounds 56 and 58, however, had activity at all four receptors. These results suggest that the 8-chloro /napthyl combination is more highly favored at the melanocortin receptors than the 8-chloro/biphenyl combination, possibly because the napthyl group is in a better position to make key ligand-receptor interactions. Two aliphatic groups were tested at th e R2 position, propyl (compound 46) and butyl (compound 47). These compounds were identical at positions R1 and R3, differing only at position R2. Additionally, the only difference between the substituents used at R2 is the addition of one methyl in compound 47. Interestingly, the propyl compound is active at all four melanocortin receptors, while the butyl com pound has only slight agonist activity at the mMC1R. N-terminal capping of the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 by Holder et al. revealed that activity increased with increasing chain length.34 This is different from the current data, where the propyl is more potent than the bu tyl. This may be due to a different mechanism

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176 of action. It was proposed that the capped tetr apeptide may have more activity because the aliphatic group associates with the plasma me mbrane, prolonging the association of the ligand with the receptor.34 The R2 position of the 1,4-benzodi azepine-2,5-diones may interact differently than the tetrapeptide. It would be in teresting to conduct furt her studies incorporating aliphatic groups of differing leng ths, both longer and shorter than those already tested. It may be that incorporation of an ethyl, methyl, or hydr ogen onto position R2 may result in greater potency. There is also the possibility that as chain length increases; the potency may gradually start to increase as well. From the R1 and R2 data, some general c onclusions may be drawn. At the mMC1R and mMC5R, the following potency series was observe d: 8-chloro/benzyl > H/benzyl > H/propyl > 8-chloro/napthyl > H/biphenyl > H/napthyl > 8-chloro/biphenyl > H/butyl > 9-methyl/benzyl. At the mMC3R and mMC4R, the potency series changes: 8-chloro/b enzyl > H/propyl > 8chloro/napthyl > H/biphenyl. Othe r combinations did not exhibit activity at the mMC3,4R. Position R3 The final position to be discussed is the R3 position. This level of diversity was added through the addition of amino esters. Thr ee different esters were used, the derivatives of phenylalanine, arginine, and ly sine (Figure 6-4). The L forms of these amino acids were used, and these were chosen based on past SAR studi es of melanocortin ligands. Both Phe and Arg have been shown to be involved in the core se quence of melanocortin lig ands. When these amino acids are changed to alanine, there is a si gnificant loss of potenc y at the melanocortin receptors.115 Lysine was chosen due to the fact th at many small molecule ligands contain a primary amine. A basic residue has been shown to be important for intera ction with a negativelycharged binding pocket in the melanocortin receptors.43,44,48-50,341,342 Arginine fulfills this requirement; however the guanidinyl side chain is large and less stable than the primary amine of

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177 lysine. It would be expected, base d on results presented in Chapter 5 of this disser tation, that the Arg compounds would be more potent th an the Lys compounds, due to a cationinteraction present in the peptide. These results are discussed below. The first series contains a hydrogen in position R1, and a benzyl in position R2. The benzyl (compound 33) and aminobutyl (compound 34) compounds were synthesized by Christine G. Joseph. From Dr. Joseph’s data it appeared that the arginine compound was more potent than the benzyl compound at the mMC1 R, though the difference was less than the standard 3-fold experimental error. When the lysine com pound (compound 39) was synthesized in this study, however, the compound only had weak stimulatory activity at the mMC1R. When R1 is a hydrogen and R2 is a biphe nyl (compounds 40-42), a different trend is observed. At the mMC1R, Phe > Arg > Lys. Additionally, at the mMC3,4R, compound 40 (H/biphenyl/Phe) has low micr omolar activity, while the other two compounds do not have any stimulatory activity. When R1 is a hydrogen and R2 is a napthyl (compounds 43-45), the potency trends change again. At the mMC1R, Arg > Lys > Phe. Ther e was no activity at the mMC3,4R, though there was activity at the mMC5R. At this receptor, there was micromolar activity when R3 was Lys (compound 45), though only slight activity was ob served with the other two compounds. Compounds 48-50 contain a 9-methyl at R1 and a benzyl at R2. These compounds had some activity at the mMC1R only. In these compounds, Lys > Arg > Phe. The Arg and Phe compounds only have weak stimulatory activ ity, but the Lys compound (compound 50) is a partial agonist with on ly 50% activity, but had a low nanomolar EC50 value. The most potent series of compounds i nvolves compound 37, which was synthesized by Dr. Joseph, along with compounds 51 and 52 synthe sized in this study. These compounds have

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178 an 8-chloro in the R1 position and a benzyl in the R2 position. What is interesting about these compounds is that 37 is a nanomolar agonist at all receptors (R3 = Lys), compound 52 is a micromolar agonist at the mMC1,3,5R (R3 = Arg) and compound 51 is a micromolar agonist at the mMC1R only (R3 = Phe). As the R3 substituent goes from Lys to Arg to Phe, the potency decreases and activity is lost at certain receptors. This may give an idea about what interactions are important at which receptor. For example, the ly sine is active at all f our receptors, but when it is changed to an arginine, activity is lost at the mMC4R and decreased at the other receptors. When a phenylalanine is introdu ced, activity is lost at the mMC3R, the mMC4R, and the mMC5R and significantly decreased at the mMC1 R. These receptor specificities may be important in designing ligands to spec ifically target certain receptors. When R1 is an 8-chloro and R2 is a biphenyl (compounds 53-55), all activity is lost at the mMC3-5R. Also, the only compound with sign ificant activity at the mMC1R is the Arg compound, 54, which is a micromolar agonist. Th e Phe compound 53 has only weak stimulatory activity at the mMC1R, and the Lys compound, 55, has no activity at any of the receptors and is cytotoxic at high concentrations. These results in dicate that the 8-chloro/biphenyl combination is not tolerated well at the melanocortin receptors and is not a good candidate for further lead optimization. The final group of compounds contains an 8-chlo ro at R1 and a napthyl at R2 (compounds 56-58). Unlike the biphenyl compounds, these co mpounds have some activity at all receptors tested. When R3 is a Phe, micromolar activit y was observed at all receptors except for the mMC4R, which had only weak stimulatory act ivity. When R3 was an Arg (compound 57), however, there was only weak activity observed at the mMC1,5R and no activity observed at the

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179 mMC3,4R. When R3 was converted to a Lys (compound 58), the molecule was a low micromolar agonist at the mMC1R, and some weak activity was observed at all other receptors. Receptor Specificities In order to draw some general conclusions from this study, it is necessary to look at specific requirements at each receptor. First, th e mMC1R will be discussed. This receptor seems to have the least strict require ments for activity. At least some stimulatory activity was seen at this receptor for all compounds tested, excep t for compound 55, which was toxic. All other combinations of building blocks tested result ed in compounds with activity, though the most potent compounds were 40 (H/biphenyl/ben zyl), 46 (H/propyl/b enzyl), 52 (8chloro/benzyl/propyl guanidine), 56 (8 -chloro/napthyl/benzyl), and 58 (8chloro/napthyl/aminobutyl). These compounds are all very different structurally, so no specific conclusions can be drawn, indicating that the local 3-D conformations of each molecule are making unique and satisfactory interactions with the receptor to result in activity. There were not many compounds tested that had activity at the mMC3R or the mMC4R. These compounds were 40 (H/biphenyl/ben zyl), 46 (H/propyl/b enzyl), 52 (8chloro/benzyl/propyl guanidine), 56 (8 -chloro/napthyl/benzyl), and 58 (8chloro/napthyl/aminobutyl). All of these compounds also had activity at the mMC1R and the mMC5R, so there was no selectivity observed for these compounds at any specific receptor. Once again there does not appear to be any spec ific pharmacophore that may be elucidated from these results, indicating specific local ligand-receptor interactions may be influencing activity for each compound. The mMC5R seems to have similar requireme nts as the mMC1R, however all ligands tested were less active at the mMC5R. The most potent compounds observed were 46

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180 (H/propyl/benzyl), 52 (8-chloro/ benzyl/propyl guanidine), and 56 (8-chloro/napthyl/benzyl). These compounds were also active at all other receptors tested. Future studies of this class of compounds should focus on the incorporation of aliphatic groups at R2, as well as halogens at R1. The R3 position seems to be less strict, allowing for the incorporation of building blocks mimicking amino acid side chains of Phe, Lys, Arg, and Trp as well as others. The His side chain cannot be inco rporated using this synthetic scheme due to the low stability of the imidazole side chain unde r the reductive environm ent necessary for its addition. It may also be interesting to incorporate substituted derivatives of Phe, as some of these have been shown to result in compounds w ith activity at the melanocortin receptors.7 In conclusion, this research has resulted in the identification of several benzodiazepine derivatives that exhibit agonist activity at the mouse melanocortin receptors. These compounds appear to have the most activity at the mMC1R, with only a few compounds exhibiting significant activity at the other receptors. These compounds are easy to synthesize due to the use of solid phase organic synthesis and may be r eadily purified allowing for the design of a large number of compounds. When substituted with build ing blocks that mimic the active sequence of peptide melanocortin ligands, this template has the potential to cr eate compounds with increasing potency that may be used in the future as drugs to treat obesity. Characterization Data Structures of compounds ma y be found in Figure 6-3. 3-(4-Aminobutyl)-1-benzyl-3,4-dihydro-1H -benzo[e][1,4]diazepine-2,5-dione (39). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxy borohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Lys(Boc)-O Me.HCl (890 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resi n containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-am inobenzoic acid (411 m g, 3.0 mmol) overnight

PAGE 181

181 and washed as before. A solution of acetanilid e (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated re sin and stirred under argon for 30 h. Benzyl bromide (1.4 ml, 12 mmol) was added following lactamization and stir red until the pH=5 as measured by pH paper. The resin was then washed and dried in a desi ccator. The compound was cleaved from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RPHPLC C18 bonded silica column (Vydac 218TP 1010, 1.0 x 25 cm) using a gradient of 22-32% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.7 7.6 min gave a compound that was >98% pure. M = 336.86 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.70 (br, 1H), 7.65—7.63 (m, 1H), 7.56—7.47 (m, 2H), 7.30—7.18 (m, 4H), 7.09—7.08 (m, 2H), 5.33 (d, J = 9.75 Hz, 1H), 4.98 (d, J = 10.0 Hz, 1H), 3.73 (br, 1H), 2.75—2.60 (m, 2H), 1.85— 1.75 (m, 1H), 1.75—1.65 (m, 1H), 1.50—1.45 (m, 2H), 1.40—1.25 (m, 2H). 3-Benzyl-1-biphenyl-2-ylmethyl-3,4-dihydro -1H-benzo[e][1,4]diazepine-2,5-dione (40). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxy borohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe.HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-am inobenzoic acid (411 m g, 3.0 mmol) overnight and washed as before. A solution of acetanilid e (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated re sin and stirred under argon for 30 h. 2-Phenylbenzylbromide (2.2 ml, 12 mmol) was added following lactamization a nd stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccator. The compound was cleaved from the

PAGE 182

182 resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218T P1010, 1.0 x 25 cm) using a gradient of 5565% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 7.1-7.9min gave a compound that was >95% pure. M = 433.1 by mass spectrometry. 1H NMR (500 MHz, DMSOd6) 8.76 (d, J = 2.6 Hz, 1H), 7.56 (dd, J = 0.6, 3.0 Hz, 1H), 7.467.37 (m, 4H), 7.29-7.15 (m, 11H), 7.077.03 (m, 2H), 5.15 (d, J = 6.4 Hz, 1H), 4.87 (d, J = 6.4 Hz, 1H), 4.01-3.97 (m, 1H), 3.14 (dd, J = 2.2,6.4 Hz, 1H), 2.94—2.89 (m, 1H). 1-(3-(1-(biphenyl-2-ylmethyl)-2,5-dioxo-2,3,4,5 -tetrahydro-1H-benzo[e][1,4]diazepin-3yl)propyl)urea (41). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Ar g(Pbf)-OMe.HCl (1321 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and wa shed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2aminobenzoic acid (411 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this so lution was then added to the acylated resin and stirred under argon for 30 h. 2-Phenylbenzylbrom ide (2.2 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. Th e compound was cleaved from th e resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 33-40% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.7-7.4min gave a compound that was >98% pure. M = 440.59 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 9.37 (br, 1H), 9.08 (br, 1H),

PAGE 183

183 7.66—7.64 (m, 1H), 7.46—7.38 (m, 4H), 7.30—7.22 (m, 5H), 7.18—7.16 (m, 1H), 7.12—7.06 (m, 2H), 6.26 (br, 2H), 5.13 (d, J = 10.0 Hz, 1H), 4.87 (d, J = 10.5 Hz, 1H), 3.76 (br, 1H), 3.03 (br, 2H), 1.83 (s, 1H), 1.78—1.68 (m, 2H), 1.56—1.44 (m, 2H). 3-(4-aminobutyl)-1-(biphenyl-2ylmethyl)-3,4-dihydro-1H-benz o[e][1,4]diazepine-2,5-dione (42). BAL resin (333 mg, 0.9 mmol/g) was mixe d with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Lys(Boc)-OMe.H Cl (890 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmo l) in NMP and 2-aminobenzoic acid (411 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and nbutyllithium (252 L, 3.0 mmol) in THF was sti rred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated resin and stir red under argon for 30 h. 2Phenylbenzylbromide (2.2 ml, 12 mmo l) was added following lactam ization and stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccator. The compound was cleaved from the resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPL C C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 32-42% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.5-7.25 min gave a compound that was >98% pure. M = 412.58 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.66 (br, 1H) 7.65 (d, J = 4.75 Hz, 1H), 7.47— 7.36 (m, 4H), 7.31—7.22 (m, 5H), 7.16—7.19 (m, 1H), 7.11—7.06 (m, 2H), 5.16 (d, J = 10.25 Hz, 1H), 4.87 (d, J = 10.0 Hz, 1H), 3.69 (br, 1H), 2.64 (t, J = 4.75 Hz, 2H), 1.86 (s, 2H), 1.78— 1.70 (m, 1H), 1.68—1.61 (m, 1H), 1.46—1.38 (m, 2H), 1.36—1.11 (m, 1H), 1.10—1.14 (m, 1H).

PAGE 184

184 3-Benzyl-1-(naphthalen-1-ylmethyl)-3,4-dihyd ro-1H-benzo[e][1,4]diazepine-2,5-dione (43). BAL resin (333 mg, 0.9 mmol/g) was mixed w ith sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-PheOMe.HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resi n containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-am inobenzoic acid (411 m g, 3.0 mmol) overnight and washed as before. A solution of acetanilid e (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated re sin and stirred under argon for 30 h. 2-(Bromomethyl)-napthalene (2.7 g, 12 mmol) was added following lactamizati on and stirred until the pH=5 as measured by pH paper. The resin was then washed and drie d in a desiccator. The compound was cleaved from the resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vyd ac 218TP1010, 1.0 x 25 cm) using a gradient of 50-60% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 7.65-8.3 min gave a compound that was >98% pure. M = 405.64 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.88 (d, J = 3.75 Hz, 1H), 7.86—7.84 (m, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.74—7.71 (m, 1H), 7.57 (d, J = 3.5 Hz, 2H), 7.53 (t, J = 3.75 Hz, 2H), 7.48—7.46 (m, 2H), 7.38 (d, J = 4.75 Hz, 2H), 7.28—7.19 (m, 5H), 5.51 (d, J = 9.5 Hz, 1H), 5.15 (d, J = 10 Hz, 1H), 4.12—4.06 (m, 1H), 3.21 (dd, J = 8.7, 4.5 Hz, 1H), 2.99 (dd, J = 8.9, 5.6 Hz, 1H). 1-(3-(1-(Naphthalen-1-ylmethyl)-2,5-dioxo-2,3 ,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3yl)propyl)urea (44). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Ar g(Pbf)-OMe.HCl (1321 mg,

PAGE 185

185 3.0 mmol) in 1% acetic acid/DMF for 2 h and wa shed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2aminobenzoic acid (411 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this so lution was then added to the acylated resin and stirred under argon for 30 h. 2-(Bromomethyl)-napt halene (2.7 g, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. Th e compound was cleaved from th e resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 30-40% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.5-7.5 mi n gave a compound that was >98% pure. M = 414.63 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.94 (d, J = 3.75 Hz, 1H), 8.45 (br, 1H), 7.86—7.83 (m, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.75—7.73 (m, 1H), 7.65—7.63 (m, 1H), 7.60 (br, 1H), 7.53—7.52 (m, 2H), 7.48—7.44 (m, 2H), 7.32 (br, 2H), 7.29—7.22 (m, 2H), 5.50 (d, J = 10.0 Hz, 1H), 5.15 (d, J = 10.0 Hz, 1H), 3.87—3.85 (m, 1H), 3.08 (br, 2H), 1.92— 1.86 (m, 1H), 1.85 (s, 1H) 1.82—1.72 (m, 1H), 1.65—1.50 (m, 2H). 3-(4-Aminobutyl)-1-(naphthalen-1-ylmethyl )-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5dione (45). BAL resin (333 mg, 0.9 mmol/g) was mixe d with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Lys(Boc)-OMe.H Cl (890 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmo l) in NMP and 2-aminobenzoic acid (411 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-

PAGE 186

186 butyllithium (252 L, 3.0 mmol) in THF was sti rred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated resin and stir red under argon for 30 h. 2(Bromomethyl)-napthalene (2.7 g, 12 mmol) was added following lactami zation and stirred until the pH=5 as measured by pH paper. The resin wa s then washed and dried in a desiccator. The compound was cleaved from the resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPL C C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 32-42% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.5-7.25 min gave a compound that was >97% pure. M = 412.58 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.70 (br, 1H), 7.83—7.75 (m, 3H), 7.64—7.60 (m, 2H), 7.64 (br, 2H), 7.48—7.46 (m 2H), 7.27—7.21 (m, 2H), 5.52 (d, J = 9.75 Hz, 1H), 5.13 (d, J = 10.0 Hz, 1H), 3.95 (br, 1H), 2.80—2.71 (m 2H), 1.89 (s, 2H), 1.88—1.79 (m, 1H), 1.79—1.70 (m, 1H), 1.58—1.46 (m, 2H), 1.57—1.39 (m, 1H), 1.39—1.32 (m, 1H). 3-Benzyl-1-propyl-3,4-dihydro-1H-ben zo[e][1,4]diazepine-2,5-dione (46). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe.HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-aminobenz oic acid (411 mg, 3.0 mm ol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated resin a nd stirred under argon for 30 h. 1-Iodopropane (1.2 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccator. The compound wa s cleaved from the resin using

PAGE 187

187 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 40-50% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.4-7.1min gave a compound that was >95% pure. M = 309.5 by mass spectrometry. 1H NMR (500 MHz, DMSOd6) 8.70 (d, J = 2.6 Hz, 1H), 7.60-7.57 (m, 2H), 7.48 (d, J = 3.2 Hz, 1H), 7.32-7.27 (m, 3H), 7.21 (t, J = 3.0 Hz, 2H), 7.17-7.14 (m, 1H), 4.25—4.19 (m, 1H), 3.88-3.84 (m, 1H), 3.61—3.57 (m, 1H), 3.13 (dd, J = 2.2, 5.6 Hz, 1H), 2.91 (dd, J = 3.6, 5.6 Hz, 1H), 1.43-1.40 (m, 1H), 1.30-1.26 (m, 1H), 0.70 (t, J = 2.8 Hz, 3H). 3-Benzyl-1-butyl-3,4-dihydro-1H-ben zo[e][1,4]diazepine-2,5-dione (47). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe.HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-aminobenzoi c acid (411 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution wa s then added to the acylated resin and stirred under argon for 30 h. 1-Iodobutane (1.4 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccato r. The compound was cleaved from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 55-65% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 4.0-4.5min gave a compound that was >95% pure. M = 323.1 by mass spectrometry. 1H NMR (500 MHz, DMSOd6) 8.69 (d, J = 2.6 Hz, 1H), 7.60-

PAGE 188

188 7.56 (m, 2H), 7.48 (d, J = 3.2 Hz, 1H), 7.32-7.26 (m, 3H), 7.22 (t, J = 2.8 Hz, 2H), 7.17—7.14 (m, 1H), 4.31—4.25 (m, 1H), 3.87—3.82 (m, 1H), 3.63—3.57 (m, 1H), 3.12 (dd, J = 2.2, 5.6 Hz, 1H), 2.90 (dd, J = 3.6, 5.6 Hz, 1H), 1.36—1.22 (m, 2H), 1.14—1.09 (m, 2H), 0.75 (t, J = 3.0 Hz, 3H). 1,3-Dibenzyl-8-methyl-3,4-dihydro-1H-b enzo[e][1,4]diazepine-2,5-dione (48). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe.HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-3-met hylbenzoic acid (453 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilid e (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated re sin and stirred under argon for 30 h. Benzyl bromide (1.4 ml, 12 mmol) was added following lactamization and stir red until the pH=5 as measured by pH paper. The resin was then washed and dried in a desi ccator. The compound was cleaved from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RPHPLC C18 bonded silica column (Vydac 218TP 1010, 1.0 x 25 cm) using a gradient of 44-54% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 8.1-8.6min gave a compound that was >95% pure. M = 369.93 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.62 (d, J = 4.0 Hz, 1H), 7.48 (dd, J = 4.5, 0.75 Hz, 1H), 7.35 (dd, J = 5.0 Hz, 1.25 Hz, 1H), 7.28—7.27 (m, 1H), 7.26—7.25 (m, 2H), 7.24—7.22 (m, 1H), 7.21—7.20 (m, 1H), 7.19—7.15 (m, 3H), 7.04—7.01 (m, 2H), 5.38 (d, J = 9.5 Hz, 1H), 4.28 (d, J = 9.25 Hz, 1H), 3.85—3.81 (m, 1H), 3.08 (dd, J = 9.0, 3.0 Hz, 1H), 2.86 (dd, J = 9.0, 5.75 Hz, 1H), 2.41 (s, 3H).

PAGE 189

189 1-(3-(1-Benzyl-9-methyl-2,5-dioxo-2,3,4,5-te trahydro-1H-benzo[e][1,4]diazepin-3yl)propyl)urea (49). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Ar g(Pbf)-OMe.HCl (1321 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and wa shed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated w ith EDC (690 mg, 3.6 mmol) in NMP and 2-amino3-methylbenzoic acid (453 mg, 3.0 mmol) overni ght and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this so lution was then added to the acylated resin and stirred under argon for 30 h. Benzyl bromid e (1.4 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. Th e compound was cleaved from th e resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 25-33% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 5.95-6.5mi n gave a compound that was >97% pure. M = 378.70 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.78 (br, 1H), 8.66 (br, 1H), 7.48 (dd, 2H, J = 4.5, 4.5 Hz), 7.36 (br, 1H), 7.32—7.28 (m, 1H), 7.16—7.15 (m, 2H), 7.2—6.8 (m, 2H), 6.25 (br, 2H), 5.39 (d, 1H, J = 9.25 Hz), 4.27 (d, 1H, J = 9.25 Hz), 3.63 (br, 1H), 3.02 (br, 2H), 2.41 (s, 3H), 1.86 (s, 1H), 1.74—1.64 (m, 2H), 1.56—1.44 (m, 2H). 3-(4-Aminobutyl)-1-benzyl-9-methyl-3,4-dihydr o-1H-benzo[e][1,4]diazepine-2,5-dione (50). BAL resin (333 mg, 0.9 mmol/g) was mixed w ith sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Lys(Boc)-OMe.HCl ( 1187 mg, 3.0 mmol) in 1% acetic acid/DMF for

PAGE 190

190 2 h and washed with DMF, DCM, and MeOH. Th e resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP an d 2-amino-3-methylbenzoic acid (453 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and nbutyllithium (252 L, 3.0 mmol) in THF was sti rred for 30 min and then 15 ml of DMF was added; this solution was then added to the acy lated resin and stirred under argon for 30 h. Benzyl bromide (1.4 ml, 12 mmol) was added following l actamization and stirred until the pH=5 as measured by pH paper. The resin was then wash ed and dried in a desiccator. The compound was cleaved from the resin using 90% TF A, 5% dimethylsulfide, and 5% H2O for 50 h. Semipreparative HPLC on an RP -HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 20-30% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 7.48.0min gave a compound that was > 95% pure. M = 350.77 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 4.48 (d, J = 3.25 Hz, 1H), 7.50—7.48 (m, 1H), 7.44 (dd, J = 4.5, 0.75 Hz, 1H), 7.32—7.28 (m, 1H), 7.19—7.14 (m, 3H), 7.02—6.99 (m, 2H), 5.38 (d, J = 9.5 Hz, 1H), 4.27 (d, J = 9.5 Hz, 1H), 3.57 (br, 1H), 3.29 (b r, 2H), 2.67—2.62 (m, 2H), 2.45—2.44 (m, 2H), 2.41 (s, 3H), 1.89 (s, 2H), 1.74—1.66 (m, 1H), 1.66—1.56 (m, 1H), 1.46—1.36 (m, 2H). 1-(3-(1-Benzyl-8-chloro-2,5-dioxo-2,3,4,5-te trahydro-1H-benzo[e][1,4]diazepin-3yl)propyl)urea (51). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and LPhe-OMe.HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated w ith EDC (690 mg, 3.6 mmol) in NMP and 2-amino4-chlorobenzoic acid (515 mg, 3.0 mmol) overni ght and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30

PAGE 191

191 min and then 15 ml of DMF was added; this solu tion was then added to the acylated resin and stirred under argon for 30 h. Benzyl bromid e (1.4 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. Th e compound was cleaved from th e resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 50-60% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.6-7.2min gave a compound that was >98% pure. M = 389.72 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.86 (d, J = 3.75 Hz, 1H), 7.62 (br, 1H), 7.56 (d, J = 5.75 Hz, 1H), 7.34—7.30 (m, 3H), 7.25 (t, J = 4.6 Hz, 4H), 7.21— 7.18 (m, 2H), 7.08 (d, J = 5.25 Hz, 2H), 5.39 (d, J = 10.0 Hz, 1H), 4.99 (d, J = 9.75 Hz, 1H), 4.14—4.06 (m, 1H), 3.16 (dd, J = 9.6, 3.0 Hz, 1H), 2.95 (dd, J = 8.75, 5.5 Hz, 1H). N-[3-(1-Benzyl-8-chloro-2,5dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl]-guanidine (52). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Ar g(Pbf)-OMe.HCl (1321 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and wa shed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated w ith EDC (690 mg, 3.6 mmol) in NMP and 2-amino4-chlorobenzoic acid (515 mg, 3.0 mmol) overni ght and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this so lution was then added to the acylated resin and stirred under argon for 30 h. Benzyl bromid e (1.4 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. Th e compound was cleaved from th e resin using 90% TFA, 5%

PAGE 192

192 dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 28-38% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.9-7.5min gave a compound that was >95% pure. M = 401.1 by mass spectrometry. 1H NMR (500 MHz, DMSOd6) 8.74 (d, J = 2. Hz, 1H), 7.64 (d, J = 3.2 Hz, 1H), 7.59 (d, J = 0.8 Hz, 1H), 7.47—7.43 (m, 1H), 7.35 (dd, J = 0.8, 3.4 Hz, 1H), 7.27—7.18 (m, 3H), 7.08 (d, J = 2.8 Hz, 2H), 6.51 (s, 1H), 5.35 (d, J = 6.4 Hz, 1H), 5.00 (d, J = 6.4 Hz, 1H), 3.88—3.84 (m, 1H), 3.10—3.06 (m, 2H), 1.89-1.80 (m, 1H), 1.72-1.63 (m, 1H), 1.60-1.45 (m, 2H). 3-Benzyl-1-(biphenyl-2-ylmethyl)-8-chloro3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5dione (53). BAL resin (333 mg, 0.9 mmol/g) was mi xed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe.HC l (647 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmo l) in NMP and 2-amino-4-chlorobenzoic acid (515 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated resin and stirred under argon for 30 h. 2-Phenylbenzylbromide (2.2 ml, 12 mmol) was added following lactami zation and stirred until the pH=5 as measured by pH paper. The resin wa s then washed and dried in a desiccator. The compound was cleaved from the resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPL C C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 65-75% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.75-7.35min gave a compound that was >98% pure. M = 465.71 by mass

PAGE 193

193 spectrometry. 1H NMR (400 MHz, DMSOd6) 8.81 (d, J = 4.0 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.46—7.37 (m, 3H), 7.30—7.25 (m, 7H), 7.23—7.21 (m, 2H), 7.18—7.16 (m, 2H), 7.11— 7.08 (m, 2H), 5.27 (d, J = 10.0 Hz, 1H), 4.83 (d, J = 10.0 Hz, 1H), 4.06—4.00 (m, 1H), 3.11 (dd, J = 8.8, 3.25 Hz, 1H), 2.90 (dd, J = 8.75, 5.75 Hz, 1H). 1-(3-(1-(Biphenyl-2-ylmethyl)-8chloro-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propyl)urea (54). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Arg(Pbf)-OMe.HCl (1321 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4-chlorobenzoic ac id (515 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution wa s then added to the acylated resin and stirred under argon for 30 h. 2-Phenylbenzylbromide (2.2 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. The compound was cleaved fro m the resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm ) using a gradient of 40-48% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 5.4-5.9 min gave a compound that was >97% pure. M = 474.59 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 9.02 (br, 1H), 8.67 (br,1H), 7.65—7.61 (m, 1H), 7.46—7.39 (m, 3H), 7.33—7.22 (m, 5H), 7.17—7.15 (m, 1H), 7.12—7.09 (m, 2H), 6.25 (br, 2H), 5.26 (d, J = 10.0 Hz, 1H), 4.81 (d, J = 10.25 Hz, 1H), 3.76 (br, 1H), 3.03 (br, 2H), 1.84 (s 1H), 1.78—1.64 (m, 2H), 1.66—1.42 (m, 2H).

PAGE 194

194 3-(4-Aminobutyl)-1-(biphenyl-2-ylmethyl)-8-c hloro-3,4-dihydro-1H-benzo[e][1,4]diazepine2,5-dione (55). BAL resin (333 mg, 0.9 mmol/g) was mi xed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Lys(Boc)-O Me.HCl (1187 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DM F, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4chlorobenzoic acid (515 mg, 3.0 mmol) overnig ht and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this so lution was then added to the acylated resin and stirred under argon for 30 h. 2-Phenylbenzylbrom ide (2.2 ml, 12 mmol) was added following lactamization and stirred until the pH=5 as meas ured by pH paper. The resin was then washed and dried in a desiccator. Th e compound was cleaved from th e resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 40-50% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 7.6-8.0min gave a compound that was >97% pure. M = 446.67 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.66 (br, 1H), 7.62 (d, J = 5.25 Hz, 1H), 7.45—7.41 (m, 3H), 7.32—7.26 (m, 5H), 7.19—7.14 (m, 1H), 7.20—7.16 (m, 2H), 5.28 (d, J = 11.0 Hz, 1H), 4.81 (d, J = 9.75 Hz, 1H), 3.95 (br, 1H), 2.59 (br, 2H), 2.14— 2.08 (m, 1H), 1.87 (s, 2H), 1.76—1.68 (m, 1H), 1.68—1.60 (m, 2H), 1.43—1.34 (m, 2H). 3-Benzyl-8-chloro-1-naphthalen-2-ylmethyl -3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5dione (56). BAL resin (333 mg, 0.9 mmol/g) was mixe d with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe.HC l (647 mg, 3.0 mmol) in 1% acetic

PAGE 195

195 acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmo l) in NMP and 2-amino-4-chlorobenzoic acid (515 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution was then added to the acylated resin and stirred under argon for 30 h. 2-(Bromomethyl)-napthalene (2.7 g, 12 mmol) wa s added following lactamization and stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccator. The compound was cleaved from the resin usi ng 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPL C C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 55-65% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 7.5-8.2min gave a compound that was >95% pure. M = 440.5 by mass spectrometry. 1H NMR (500 MHz, DMSOd6) 8.89 (d, J = 2.6 Hz, 1H), 7.85-7.84 (m, 1H), 7.80 (d, J = 3.4 Hz, 1H), 7.73-7.72 (m, 1H), 7.65 (d, J = 0.6 Hz, 1H), 7.57—7.54 (m, 2H), 7.50—7.45 (m, 2H), 7.35—7.33 (m, 2H), 7.30—7.24 (m, 3H), 7.22—7.18 (m, 2H), 5.55 (d, J = 6.6 Hz, 1H), 5.14 (d, J = 6.4 Hz, 1H), 4.17-4.13 (m, 1H), 3.20 (dd, J = 2.2, 5.6 Hz, 1H), 2.98 (dd, J = 3.4, 5.0 Hz, 1H). 1-(3-(8-Chloro-1-(naphthalen-1-ylme thyl)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propyl)urea (57). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and H-Arg(Pbf)-OMe.HCl (1321 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4-chlorobenzoic ac id (515 mg, 3.0 mmol) overnight and washed as before. A

PAGE 196

196 solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution wa s then added to the acylated resin and stirred under argon for 30 h. 2-(Bromomethyl)-napthalene (2.7 g, 12 mmol) was added following lactamization and stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccator. The compound wa s cleaved from the resin using 90% TFA, 5% triisopropylsilane, and 5% H2O for 50 h. Semi-preparative HPLC on an RPHPLC C18 bonded silica column (Vydac 218TP 1010, 1.0 x 25 cm) using a gradient of 34-44% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 7.0-7.8min gave a compound that was >95% pure. M = 448.63 by mass spectrometry. 1H NMR (400 MHz, DMSOd6) 8.89 (br, 1H), 8.85 (br, 1H), 7.98 (br, 1H), 7.84—7.80 (m, 2H), 7.75 (d, J = 4.0 Hz, 1H), 7.66-7.60 (m, 3H), 7.48—7.47 (m, 1H), 7.34—7.32 (m, 1H), 7.21 (d, J = 5.75 Hz, 2H), 6.18 (br, 2H), 5.55 (d, J = 10.25 Hz, 1H), 5.15 (d, J = 10.25 Hz, 1H), 3.98—3.89 (m, 1H), 3.29 (br, 3H), 2.12—2.08 (m, 1H), 1.88 (s, 1H), 1.88—1.60 (m, 2H), 1.60—1.50 (m, 1H). 3-(4-Amino-butyl)-8-chloro-1-napht halen-2-ylmethyl-3,4-dihydro-1Hbenzo[e][1,4]diazepine-2,5-dione (58). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Lys(Boc)-OMe.HCl (890 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The resin containing the amino ester was then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4-chlorobenzoic ac id (515 mg, 3.0 mmol) overnight and washed as before. A solution of acetanilide (973 mg, 7.2 mmol) and n-butyllithium (252 L, 3.0 mmol) in THF was stirred for 30 min and then 15 ml of DMF was added; this solution wa s then added to the acylated resin and stirred under argon for 30 h. 2-(Bromomethyl)-napthalene (2.7 g, 12 mmol)

PAGE 197

197 was added following lactamization and stirred until the pH=5 as measured by pH paper. The resin was then washed and dried in a desiccator. The compound wa s cleaved from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semi-preparative HPLC on an RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm) using a gradient of 35-45% acetonitrile in 0.1% TFA/H2O over 10 min and collected from 6.0-6.8min gave a compound that was >95% pure. M = 422.5 by mass spectrometry. 1H NMR (500 MHz, DMSOd6) 8.75 (br, 1H), 7.86-7.84 (m, 1H), 7.80 (d, J = 3.4 Hz, 1H), 7.76—7.74 (m, 1H), 7.68 (d, J = 0.8 Hz, 1H), 7.63—7.59 (m, 2H), 7.49—7.46 (m, 2H), 7.33 (dd, J = 0.8, 3.4 Hz, 1H), 7.20 (dd, J = 0.6, 3.4 Hz, 1H), 6.55 (br, 1H), 5.57 (d, J = 6.4 Hz, 1H), 5.13 (d, J = 6.4 Hz, 1H), 3.84 (br, 1H), 2.72 (t, J = 2.8 Hz, 2H), 1.85—1.78 (m, 1H), 1.77—1.68 (m, 1H), 1.56—1.28 (m, 4H).

PAGE 198

198 CHAPTER 7 CONSTRUCTION AND CHARACTERIZATION OF MELANOCORTIN-2 AND -4 RECEPTOR CHIMERAS All primer design, cloning, stable cell lin e generation, and pharmacology was performed by Krista Wilson. Dr. Xhimin Xiang and Bettina Proneth of the Haskell-Luevano laboratory provided valuable advice and training in clon ing and pharmacology. The Flag tag was added to the hMC4R by Dr. Xhimin Xiang. Dr. Sally Lither land of the Department of Pathology provided training and data analysis advi ce for FACS analysis. Data anal ysis was performed by Krista Wilson and Dr. Carrie Haskell-Luevano. Luciferase Assay was performed with help from Dr. Hendrik Luesch. Introduction The Melanocortin-2 Receptor (MC2R), also known as the ACTH Receptor, or ACTHR, is unique among the melanocortin receptors.59 It is one of the shortest known GPCRs, with only 297 amino acids and a predicted mass of 33 kDa.53 It also has two glycosylation sites near the Nterminus, which results in an experimental mass of 43 kDa.129 The human MC2R shares 39% sequence identity with the MC1R, 45% identity with the MC3R, 50% identity with the MC4R, and 46% sequence identity with the MC5R. These differences ar e mainly in the C-terminal domain as well as the first extracellular loop a nd the third intracellular loop. The MC2R aligned with all four melanocortin receptors is presen ted in Figure 7-1. The MC2R is coded by the ACTHR gene located on the sma ll arm of chromosome 18 (18p11.21).348 The coding sequence is contained in one exon, exon 2, though a major tran scription initiation si te has been located upstream of exon 2.53 The MC2R is expressed in the adrenal cortex,53 though it has also been observed at low levels in human skin,349 ovarian steroid cell tumors,350 and in rodent adipocytes.351

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199 N-Terminus (EC) hMC1R MSSVVEFTMAVQGSQRRLLGSLNSTPTAIPQLGLAANQTGARCLEVSIS hMC2R MKHIINSYENINNTARNNSDCPRVVLP hMC3RMSSVVEFTMSIQKTYLEGDFVFPVSSSSFLRTLLEPQLGSALLTAMNASCCLPSVQPTLPNGSEHLQAPFFSNQSSSAFCEQVFIK hMC4R MVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS hMC5R MSSFHLHFLDLNLNATEGNLSGPNVKNKSSPCEDMGIA TM1 Loop 1 (IC) hMC1R DGLFLSLGLVSLVENALVVATIAK NRNLHS hMC2R EEIFFTISIVGVLENLIVLLAVFK NKNLQA hMC3R PEVFLSLGIVSLLENILVILAVVR NGNLHS hMC4R PEVFVTLGVISLLENILVIVAIAK NKNLHS hMC5R VEVFLTLGVISLLENILVIGAIVK NKNLHS TM2 Loop 2 (EC) hMC1R PMYCFICCLALSDLLVSGSNVLETAVILL LEAGALVARAAVLQQL hMC2R PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA hMC3R PMYFFLCSLAVADMLVSVSNALETIMIAIVHSDY LT FED QFIQHM hMC4R PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI hMC5R PMYFFVCSLAVADMLVSMSSAWETITIYL LNNKHLVIADAFVRHI TM3 Loop 3(IC) hMC1R DNVIDVITCSSMLSSLCFLGAIAVDRYISI FYALRYHSIVT hMC2R DDIIDSLFVLSLLGSIFSLSVIAADRYITI FHALRYHSIVT hMC3R DNIFDSMICISLVASICNLLAIAVDRYVTI FYALRYHSIMT hMC4R DNVIDSVICSSLLASICSLLSIAVDRYFTI FYALQYHNIMT hMC5R DNVFDSMICISVVASMCSLLAIAVDRYVTI FYALRYHHIMT TM4 Loop 4 (EC) hMC1R LPRARRAVAAIWVASVVFSTLFI AYYDH hMC2R MRRTVVVLTVIWTFCTGTGITMV IFSHH hMC3R VRKALTLIVAIWVCCGVCG V V FIVYS hMC4R VKRVGIIISCIWAACTVSGILFI IYSDS hMC5R ARRSGAIIAGIWAFCTGCGIVFI LYSES TM5 Loop 5 (IC) hMC1R VA VLLCLVVFFLAMLVLMAVLYVHMLARACQHAQG IARLHKRQRPVHRPVHQ hMC2R VPT VITFTSL FPLMLVFILCLYVHMFLLARSHTRK ISTLP hMC3R ESKMVIVCLITMFFAMMLLMGTLYVHMFLFARLHVKR IAALPPADGVAPQQH hMC4R SA VIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ hMC5R TY VILCLISMFFAMLFLLVSLYIHMFLLARTHVKR IAALPGASSARQRTSMQ TM6 Loop 6(EC) hMC1R GFGLKGAVTLTILLGIFFLCWGPFFLHLTLIVL CPEHPTCGC hMC2R RANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC hMC3R SCMKGAVTITILLGVFIFCWAPFFLHLVLIIT CPTNPYCIC hMC4R GANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC hMC5R GAVT VTMLLGVFTVCWAPFFLHLTLMLS CPQNLYCSR TM7 C-terminus (IC) hMC1R IFKNFNLFLALIICNAIIDPLIYAFHSQ ELRRTLKEVLTCSW hMC2R YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW hMC3R YTAHFNTYLVLIMCNSVIDPLIYAFRSL ELRNTFREILCGCNGMNLG hMC4R FMSHFNLYLILIMCNDIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY hMC5R FMSHFNMYLILIMCNSVMDPLIYAFRSQ EMRKTFKEIICCRGFRIACSFPRRD Figure 7-1. Amino acid alignment of human melanocortin receptors 1-5, arranged by transmembrane domain and loop. IC = Intracellular. EC = Extracellular. Transmembrane (TM) domains were determined by comparison with previous hMC1R and hMC4R alignments.43 Spaces are placed in sequences for alignment purposes.

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200 The most unique aspect of the MC2R is its response to the endogenous melanocortin ligands. The MC1,3-5R all respond to the melanoc yte stimulating hormones (MSH) as well as ACTH. The MC2R, however, only responds to ACTH and not to the MSH ligands.59 The mechanism behind this ligand selectivity is not known as it has been historically difficult to express the MC2R in non-adrenal cell lines, making it difficult to a ssay new compounds. The ligand selectivity of the MC2R is an interesting area of exploration, as -MSH represents the first thirteen amino acids of ACTH (Figure 7-2) It has been proposed that two ligand domains are required for ligand binding to the MC2R.59,131,352,353 It not only requires the known His-PheArg-Trp tetrapeptide that is the minimum seque nce required for nM activity at the other melanocortin receptors, it also requ ires a basic region present at th e C-terminus of ACTH that is not present in the MSH peptides.59,131,352,353 Another unique aspect of th e MC2R is that it is not expressed in cells of non-adrenal origin.59 It has been shown that cell lines of adrenal origin such as Y6 and OS3 are the only cell types able to express a functional MC2R when the receptor is stably transfected into the cell lines.59 These cell lines were derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line and contai n a wild type MC2R gene, however this DNA sequence is not functional.228 Non-transfected OS3 cells do not express the MC2R gene as is reported here in real-time PCR experiments (Fi gure 7-6). When these OS3 cells are transfected with the hMC2R gene, a functional protein is ex pressed on the cell surfac e as shown by real-time PCR, the cAMP functional assay, and FACS (Fluor escence-Activated Cell So rting) (Figure 7-5). Figure 7-2. -MSH is formed from the e ndogenous truncation of ACTH.

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201 Figure 7-3. Schematic of chimeras. Blue cylin ders = hMC4R TM domains; Red cylinders = hMC2R TM domains.

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202 Cell lines of non-adrenal origin can be stably transfected with the re ceptor, but there is no functional response when stimulated with ACTH.59 HEK293 cells are derived from human embryonic kidney cells. These cells are not of adrenal origin and do not contain the MC2R gene. Until this time, it was assumed that the reason the MC2R did not produce a functional response when expressed in HEK293 cells was due to the fact that the receptor was not expressed on the cell surface.86 This work demonstrates that this may not be the case and the MC2R is expressed on the surface of HEK293 cells as determined by FA CS (Figure 7-5C). It is hypothesized that the reason MC2R-HEK cells do not produ ce a functional response in the -galactosidase assay is due to lack of an accessory protein which binds to the receptor and ligand, facilitating receptor stimulation. Alternatively, the MC2R is in an inactive conformation and thus is not able to bind the ligand. These results lead to the idea that an a dditional factor may be needed to produce a functional MC2 receptor. The OS3 cells naturally express this receptor, therefore they would also express all proteins required for activit y. HEK293 cells do not express this receptor naturally; therefore they may not express this unknown factor. Two classes of accessory proteins have gained popularity in receptor interaction. The first is a class known as RAMPs, or receptor activity modifying proteins, which have been identif ied as essential partners for the activity at the calcitonin receptor (CTR) and the cal citonin receptor-like receptor (CRLR).354 There are three RAMPs, RAMP1-3, that have been identified to date. RAMP 1 is a 148 amino acid protein which shares only 30% homol ogy with the other two RAMPs.354 All RAMPs share the same structure. They are made up of a large extracel lular N-terminus, a single TM domain and a short intracellular C-terminal domain.354 They have been shown to form dimers with their target receptors in the endoplasmic reticulum and rema in associated through the Golgi apparatus,

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203 during insertion into the cell membrane, and through receptor activation, internalization and degradation.355-359 Additionally, RAMP association l eads to glycosylation of the CRLR354,357 and plays a role in ligand-receptor binding as proposed by Foord et al .360 Due to its ubiquitous expression,354 it has been suggested that RAMPs may play a role in the function of other receptors. MRAP, or Melanocortin-2 Receptor A ccessory Protein, was first identified as fat tissue-specific low molecular weight protein (F alp) expressed in adipocytes and the adrenal cortex.361 Metherell et al. has identified that MRAP interacts with the MC2R and is required for MC2R activity in CHO cells.86 The protein has been shown to be expressed in 3T3-L1 cells361 as well as Y1 and Y6 cells,86 all which express a functional MC2R. Recently, it has been shown that the MC2R is expressed on the surface of HEK293 cells without the presence of MRAP, but that cotransfection of MRAP is required for MC2R activity in this cell line.362 For many years ligands have been screened at the melanocortin receptors using the galactosidase assay.209 This assay works well with receptors expressed in HEK293 cells and the pCRE/ -gal plasmid transiently transfected into the cells before stimulation.209 This method is effective for cells expressing the MC1R, MC3R, MC4R, or MC5R, however, when the MC2R is stably transfected into HEK 293 cells, th ere is no functional response in the -galactosidase assay.83 As has been previously stated, it is known that stimulation of OS3 cells stably transfected with the hMC2R gene results in a functional response.59 Conversely, HEK293 cells that contain the hMC2R do not produce a functiona l response when stimulated.84,85 Amino acid alignment of the hMC2R with the hMC4R has identified ke y amino acid changes in the transmembrane regions which may be involved in ligand binding a nd/or signal transduction (Figure 7-1). To test the hypothesis that these amino acids are involv ed in the production of a functional hMC2

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204 receptor in HEK293 cells, chimeric receptors were made to identify the role each transmembrane domain plays in ligand binding and signal tran sduction in the hMC2R. Entire transmembrane regions from the hMC4R were inserted into the hMC2R gene and these chimeric receptors were stably transfected into OS3 ce lls and HEK293 cells. Figure 7-3 de picts the chimeric receptors that were generated. Based on the regions of diversity in the transmembrane domains, hMC2R chimeras were created in which TM domain s 2, 5, and 7 from the hMC4R replace the hMC2R sequence. Chimeras were also generated which contain the N-terminus, C-terminus, and middle three TM domains of the hMC4R, respectively. A site-directed mutagenesis approach will not be used because the differences may not be due to single amino acid differences, but due to the local tertiary structure of all amino acids in the vi cinity. Identical chimeras in which hMC2R TM domains are inserted into the hMC4R were also generated to serve as internal controls. All chimeras were stably expressed in both the OS3 and HEK293 cells. The wild type receptors were also expressed in both cell types as controls. Figure 7-3 depicts the chimeras that were made. Blue TM domains are used to represent the sect ions from the hMC4R, and red TM domains are used to depict regions of the hMC2R. Following construction of chimer as and generation of stable cell lines, the expression and localization of the receptor was determined using FACS (Fluorescence-Activated Cell Sorting). All receptors contain the Flag tag on the N-terminus, as demonstrated by our lab.363,364 A Flag tag is an amino acid sequence specific for an anti -Flag antibody that is a ttached to the receptor by PCR. When labeled with the anti-Flag-AP C antibody, cells fluoresce, allowing the flow cytometer to determine relative fluorescence leve ls. Cells were first la beled on the extracellular surface, and then a group of cells were permeabili zed and labeled again, to label the receptor on the inside of the cell. These ce lls are used to determine the to tal receptor expression. The cells

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205 labeled only on the outside give cell surface expre ssion. Relative surface expression of chimeras was determined based on relative to surface expr ession of the wild type receptors. Functional assay of all chimeras in HEK293 cells was al so performed to measure the response of the receptor to agonist stimulation. Results Flag-tagged hMC2R A Flag tag was added to the N-terminus of the hMC2R to enable detection with an antibody specific for the Flag sequence, DYKDDDDK.365 In order to insert the tag into the receptor sequence, a primer was designed that c ontained the Kozak sequence, which is necessary for ribosome recognition, a Met start codon, and th e DNA sequence that codes for the Flag tag (Figure 7-4 and Table 7-1). The hMC2R gene w ith the new 5’ sequence was amplified by PCR, then the new DNA was isolated and gel purified. During PCR, the HindIII restriction site was inserted at the 5’ end and the Xba1 restriction s ite inserted at the 3’ e nd. Both the insert and a new pBKS or pcDNA3 plasmid were cut with these two enzy mes, and the insert ligated into the pBKS or pcDNA3 plasmid. This resulted in a plasmid containing the Flag-hMC2R construct. Figure 7-4. Addition of Flag tag to the hMC2R gene. A PCR approach wa s used to create a receptor preceeded by the Kozak sequence, Met start codon, and Flag tag, and then the construct was ligated in to a new expression plasmid. This construct was stably transfected into bot h HEK293 and OS3 cells in order to test the surface expression and functional activity of the receptor. First, FACS analysis and deconvolution microscopy were used to test the surface expression of the receptor. One group of

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206 cells was labeled with an an ti-Flag antibody on the cell surface only, and another group was labeled on the outside and the inside of the ce ll. Flow cytometry allowed quantification of surface expression of the Flag-hMC2R in both cell types. Results are shown in Figure 7-5A and B. Figure 7-5A shows the percentage of cells expressing the receptor on the surface and in total above background levels. Figure 7-5B shows the mean fluorescence of each cell type and location above background. The mean fluorescen ce gives information about the amount of receptor in each cell. A high mean fluorescence indicates that individual cells are expressing large numbers of receptor while a low mean fluorescence indicates that individual cells are expressing low levels of receptors. Visualizatio n of the surface expression of Flag-hMC2R was accomplished by deconvolution microscopy. Figure 7-5C shows an image of an HEK293 cell expressing the Flag-hMC2R and labeled on the ce ll surface only. Figure 7-5D shows the surface expression of the Flag-hMC2R in OS3 cells. It is apparent that the HEK cells express the receptor at much higher levels than OS3 cells, t hough much of the receptor was contained in the interior of the cell. OS3 cells have been shown to have low transfection efficiency, which is apparent here, but most of the receptor that is expressed is on the surface as opposed to the interior of the cell. Table 7-1. Primers for insertion of Flag tag into hMC2R template. F = forward primer; R = reverse primer. Primer Primer Sequence Flag-hMC2R-F 5’GATGAAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACAT TATCAACTCG3’ Flag-hMC2R-R 5’CGAGGCTGATCAGCGGGTTTAAACG3’ The Flag-hMC2R was also iden tified by Western blotting of the transfected HEK and OS3 cell lines. These results are shown in Figure 7-5E. Treatment of ce llular extracts with an antiFlag antibody reveals the presence of the Flag-tagged receptor in the cell.

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207 Figure 7-5. Characterization of HEK293 and OS3 cells stably e xpressing the Flag-hMC2R. A. Surface and total expression levels of Fl ag-hMC2R determined by FACS. B. Mean fluorescence levels of surface and total Flag-hMC2R expression in HEK and OS3 cells. C. Deconvolution microscopy image of Flag-hMC2R stably expressed in HEK cells and labeled with anti-Flag-APC antibody on the cell surface only. D. Deconvolution microscopy image of Flag-hMC2R stably expressed in OS3 cells and labeled with anti-Flag-APC antibody on th e cell surface only. E. Western blot of HEK and OS3 cells stably expressing Fl ag-hMC2R. F. cAMP production determined by cAMP assay in OS3 cells stably expre ssing the Flag-hMC2R (cells obtained from Schimmer et al .) and stimulated with ACTH(1-24). Functional activity of the receptor in each ce ll type was tested using the cAMP assay. Results are shown in Figure 7-5F. As has been previously observed by other researchers, a

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208 functional response was seen in OS3 cells expr essing the Flag-hMC2R and stimulated with ACTH, however; no functional activity was obs erved in the HEK cells (data not shown). Real-time PCR Real-time PCR was performed in order to determine if the accessory proteins RAMP1, RAMP2, RAMP3, or MRAP were present in the HEK and OS3 cells. Probes for the hMC2R as well as the four accessory proteins were purchas ed from Applied Biosciences. Expression levels were tested in five cell types: hMC2R-OS3 cells, hMC4R-HEK cells, untransfected OS3 cells, and the hMC2R-OS3 and hMC4R-HEK cells stimulated with 10-7M ACTH(1-24) for 6 h at 37oC. The hMC2R gene expressed in transfected OS3 cells was used as a 100% standard. All values were normalized to 18S ribosomal RNA levels. Each experiment was performed at least three times with at least tw o replicates per experiment. Figure 7-6. Gene expression a ssay of hMC2R-OS3, hMC4R-HEK, and OS3 cells. Stimulated cell lines were treated with 10-7M ACTH(1-24) for 6 h pr ior to mRNA isolation. Results are shown in Figure 7-6. OS3 cells tr ansfected with the hMC2R showed expression of hMC2R mRNA, but no other te st mRNA was detected. The stimulated hMC2R-OS3 cells

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209 showed an increase in hMC2R expression, but no other mRNA was detected. HEK cells transfected with the hMC4R gene but not the hMC2R surprisingly showed high levels of hMC2R expression. This may be explained by cross-reac tion of the hMC2R probe with the hMC4R gene. Though the probe sequence is prop rietary, it was explained by th e company that cross-reaction may occur in some instances. The hMC4R-HEK cell line also showed significant levels of the RAMP2 and RAMP3 mRNA, but no RAMP1 or MRAP. The stimulated hMC4R-HEK cells showed an upregulation of the melanocortin receptor, but decreased levels of RAMP2, and no RAMP1, RAMP3, or MRAP. Untransfected OS3 cells did not express any of the test genes. No MRAP mRNA was found in th e OS3 cells because a probe for the human MRAP (hMRAP, GenBank AF454915) was used and the OS3 cell line is a murine cell line. Figure 7-7 shows DNA base alignment between the hMRAP gene (GenBank AF454915) and the mouse Mrap (mMrap, GenBank NM_029844) gene. The sequences differ too much for the human probe to recognize the mouse gene. hMRAP 179 GCCACAGACATGGCCAACGGGACCAACGCCTCTGCCCCATACTACAGCTATGAATACTAC 238 ||||||| |||||||||||||||| ||||||||| ||| | ||||||||| || ||| mMrap 232 GCCACAGTCATGGCCAACGGGACCGACGCCTCTGTCCCGCTCACCAGCTATGAGTATTAC 291 hMRAP 239 CTGGACTATCTGGACCTCATTCCCGTGGACGAGAAGAAGCTGAAAGCCCACAAACATTCC 298 |||||||| | ||||||||||| |||||||||||||||||||||||| |||| |||||| mMrap 292 CTGGACTACATAGACCTCATTCCTGTGGACGAGAAGAAGCTGAAAGCCAACAAGCATTCC 351 hMRAP 299 ATCGTGATCGCATTCTGGGTGAGCCTGGCTGCCTTCGTGGTGCTGCTCTTCCTCATCTTG 358 || || ||||| | ||| ||||||||||| ||||||||||||| ||||| |||||| || mMrap 352 ATTGTCATCGCCCTGTGGTTGAGCCTGGCTACCTTCGTGGTGCTCCTCTTTCTCATCCTG 411 hMRAP 359 CTCTACATGTCCTGGTCCGCCTCCCCGCAGATGAGGAACAGCCCCAAGCACCACCAAACA 418 ||||||||||||||||| | |||||| ||||||||| |||| ||| | | ||| | || | mMrap 412 CTCTACATGTCCTGGTCGGGCTCCCCACAGATGAGGCACAGTCCCCAACCCCAGCCAATA 471 hMRAP 419 TGCCCCTGGAGTCACGGCCTCAACCTCC 446 || | |||| |||| || ||||||||| mMrap 472 TGTTCATGGACTCACAGCTTCAACCTCC 499 Figure 7-7. DNA alignment between hMRAP and mMrap genes.

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210 Chimera Construction Chimeras were constructed using the followi ng methods (Figure 7-8). Unique restriction sites were inserted into the w ild type DNA using PCR (Figure 7-9) Care was used not to change the amino acid sequence. Following PCR, the DNA wa s incubated with DpnI to remove the wild type DNA, and then the DNA transformed into chemically competent DH5 cells. Transformed bacteria were grown and DNA isol ated via Miniprep and sequenced to confirm the presence of the unique restriction site. The bacteria was then grown for Maxiprep and DNA isolated and stored in TE Light buffer at a concentration of 1g/L. Enzymatic digestion of the plasmid allowed for the isolation of a short insert and a digested plasmid, which were isolated via gel extraction. At this point, the insert from th e hMC4R was ligated into the digested hMC2R plasmid, and the hMC2R insert ligated into the hMC4R plasmid, resulting in chimeric receptors. The ligated DNA was transformed into chemically competent DH5 cells, and amplified by miniprep and maxiprep. The chimeric receptors were fully sequenced to ensure that the receptors were free of unintentional PCR-induced changes. Full sequences of all wild type receptors and chimeras as well as restriction ma ps may be found in Appendix B. Following chimera construction, the DNA was stab ly transfected into either HEK or OS3 cells. For HEK cells, 10 g of DNA was transfec ted using the calcium chloride precipitation method.227 For OS3 cells, 10 g of DNA was transfect ed using Fugene 6 transfection reagent (Roche). Stably transfected cells we re selected using the antibiotic G418.

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211 Figure 7-8. Schematic of chimera construction. Chim era E is used as example, BlpI and HpaI refer to restriction endon ucleases. Actual experiments performed on plasmid DNA containing melanocortin receptor DNA, data presented here as protein for clarity. DNA fragments ligated using T4 ligase. Figure 7-9. Diagram of unique rest riction sites used during this study and their locations in the receptor.

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212 Table 7-2. Restriction enzyme recognition sites. Indicates site of DNA digestion. Restriction Enzyme Recognition Sequence Hind3 5’…A AGCTT…3’ 3’…TTCGA A…5’ Blp1 5’…GC TNAGC…3’ 3’…CGANT CG…5’ Age1 5’…A CCGGT…3’ 3’…TGGCC A…5’ BsrG1 5’…T GTACA…3’ 3’…ACATG T…5’ Hpa1 5’…GTT AAC…3’ 3’…CAA TTG…5’ Xba1 5’…T CTAGA…3’ 3’…AGATC T…5’ Table 7-3. Primers for insertion of unique restri ction sites in hMC2R and hMC4R templates. F = forward primer; R = reverse primer Restriction site s are underlined. Primer Primer Sequence hMC2R-Blp1-F 5’CATCTGTAGCTTAGC CATATCTGATATGCTGGGC3’ hMC2R-Blp1-R 5’GCCCAGC ATATCAGATATGGCTAAGC TACAGATG3’ hMC2R-Age1-F 5’CCTGTCTGTGATTGCTGCGGACCGGT ACATCACC3’ hMC2R-Age1-R 5’GGTGATGTACCGGT CCGCAGCAATCACAGACAGG3’ hMC2R-BsrG1-F 5’CCT GTGCCTCTATGTACA CATGTTCCTGCTGGC3’ hMC2R-BsrG1-R 5’G CCAGCAGGAACATGTGTACA TAGAGGCACAGG3’ hMC2R-Hpa1-F 5’CATGAAAGGGGCCATCACGTTAAC CATCCTGCTCG5’ hMC2R-Hpa1-R 5’CGAGCAGGATGGTTAAC GTGATGGCCCCTTTCATG3’ hMC4R-Blp1-F 5’CTTTTTCATCTGCAGCTTAGC TGTGGCTGATATGC3’ hMC4R-Blp1-R 5’GCATATCAGCCACAGCTAAGC TGCAGATGAAAAAG3’ hMC4R-Age1-F 5’GCTTTCAATTGCAGTGGACCGGT ACTTTACTATCTTC3’ hMC4R-Age1-R 5’GAAGATAGTAAAGTACCGGT CCACTGCAATTGAAAGC3’ hMC4R-BsrG1-F 5’CATGGCTTCTCTCTATGTACA CATGTTCCTGATGG3’ hMC4R-BsrG1-R 5’CCA TCAGGAACATGTGTACA TAGAGAGAAGCCATG3’ hMC4R-Hpa1-F 5’GAAGGGAGCGATTACGTTAAC CATCCTGATTGG3’ hMC4R-Hpa1-R 5’CCAATCAGGATGGTTAAC GTAATCGCTCCCTTC3’ Fluorescence Activated Cell Sorting Fluorescence-Activated Cell Sorting was used to determine the cellula r localization of each chimeric receptor. For each cell type, four sample s were prepared (Figure 7-10). The first sample was not labeled with any antibody, and the second sample was labeled with an isotype control. This control was used to determine backgr ound fluorescence, which was subtracted from the experimental samples. The third sample was la beled with an Anti-Flag -APC antibody on the cell

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213 surface only. The fourth sample was labeled on the surface, then permeabilized with saponin and labeled again with the Anti-Flag antibody. The f ourth sample gave total expression of the receptor on the outside and inside of the cell. Th ree independent experiments were performed for each cell type. Figure 7-10. Schematic of FACS experiment. Ce lls are split into f our groups as shown. Results are shown in Figures 7-11 and 7-12. Fi gure 7-11 depicts expression data from the HEK cells expressing the chimeras. Data is repr esented in several ways. Figure 7-11A gives the raw data obtained for each cell type. Figure 7-11 B shows the data normalized to the wild type receptor. From this data, it was observed that chimeras 2C1, 2C3, and 2C6 all exhibited increased surface expression as compared to the hMC2R wild type receptor, though total expression remained the same. Additionally, ch imera 2C5 showed decr eased surface and total expression as compared to wild type. When the hMC4R chimeras were examined, it was observed that most chimeras exhibited simila r expression to the hMC4 R wild type, though 4C1 and 4C4 showed slightly less surface expression.

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214 Figure 7-11C and 7-11D show the mean fluorescence data from the HEK FACS experiments. The mean fluorescence (MF) is a measure of the relative fluorescence on each individual cell. Looking at the MF normalized to the wild type receptors, it was seen that chimeras 2C1, 2C2, and 2C6 had increased su rface and total expression on each cell, and chimera 2C3 expressed similar total levels of receptor, however the surface density was significantly increased over wild type. Figure 7-11. Summary of FACS data of wild t ype receptors and chimeras expressed in HEK293 cells.

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215 Figure 7-12. Summary of FACS data of wild type receptors and chimeras expressed in OS3 cells. FACS analysis was also performed on OS 3 cells. Figure 7-12 depicts this data. One significant observation is that th e receptor expression and mean fluorescence were significantly decreased as compared to HEK cells (Figure 7-11A and Figure 7-12A). Figure 7-12A and 7-12B shows raw and normalized FACS data in OS3 cells Similar to the HEK cells, chimeras 2C1 and 2C3 exhibit increased surface expression as comp ared to the hMC2R wild type receptor, though total expression remains fairly constant. hM C4R chimera expression was more variable. Chimeras 4C2 and 4C5 showed increased surfac e and total expression over wild type, while

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216 chimeras 4C1 and 4C4 have decreased expres sion. Chimeras 4C3 and 4C6 showed similar expression to the wild type hMC4R receptor. Figure 7-13. Histograms of FACS data from thr Flag-hMC4R construct ex pressed in both OS3 (top) and HEK (bottom) cell lines. Purple (filled) peak represents cells not labeled with antibody. Green curve represents isotype control. Blue peak represents total expression and pink peak represents surf ace expression. M1 = No label and isotype control peaks. M2 = Surface and total e xpression peaks only. M3 = Surface and total expression peaks + low data. M4 = Surface and total expression peaks + high and low data. OS3 mean fluorescence levels are shown in Fi gures 7-12C and 7-12D. While relative total expression remained the same among chimeras 2C1, 2C2, 2C3, and 2C4, surface expression was significantly less in chimeras 2C1, 2C2, 2C3, and 2C6, and total expression was decreased in

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217 chimera 2C6 as compared to hMC2R wild type. Among the hMC4R chimeras, 4C4 and 4C6 had decreased expression while the other chimeras had similar expression to the wild type hMC4R. FACS data expressed as histograms for all w ild type receptors and chimeras in both HEK and OS3 cells may be found in Appendix C. Figur e 7-13 depicts FACS hi stograms of the FlaghMC4R construct expressed in both OS3 and HEK ce ll lines. It appears th at there may be two populations of cells present, a high-affinity binding population and a lower-affinity population. It is unknown whether or not this tr end affects functional activity. Luciferase Assay Functional assay was performed on the chimeras The luciferase assay is a reporter gene assay utilizing the firefly luciferase gene231 coupled to 16 CRE (cAMP response element) units. 83,236,238,366-368 When the receptor is stimulated by an agonist, cAMP is produced, which binds CREB (cAMP response element binding protein), which binds to the CRE units, initiating transcription of the luciferase gene. Following stimulation, D-luciferin is added to the lysis mixture, which reacts with the luciferase producing a flash of light Luminescence can be quantified using a luminometer. The more lumi nescence detected, the more potent the agonist. It has been reported that the luciferase a ssay may be used with both HEK and OS3 cell types.238 However, after extensive te sting on the cell lines generate d in this study, no luciferase activity was observed in OS3 cells either with ag onist stimulation or with forskolin stimulation, which increases cAMP levels without receptor involvement. Therefore th e luciferase assay was performed only on HEK chimeras us ing the agonists ACTH(1-24) and -MSH.

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218 Figure 7-14. Summary of functiona l data from the luciferase assay performed on hMC2R wild type receptors and chimeras expressed in HEK293 cells.

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219 Figure 7-15. Summary of functiona l data from the luciferase assay performed on hMC4R wild type receptors and chimeras expressed in HEK293 cells.

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220 Functional data is shown in Figures 7-14 a nd 7-15, and in Table 7-4. Both wild type receptors exhibited agonist EC50 values within repor ted values. Most chimeras, however, did not have any activity when stimulated with ACTH(1-24) or -MSH. These inactive receptors were 2C1, 2C2, 2C3, 2C4, 2C6, 4C2, 4C4, 4C5, and 4C6. Only three chimeras had activity with these ligands: 2C5, 4C1, and 4C3. Chimera 4C1 was the most potent, with only 6-fold decreased potency as compared to the hMC4R. Like the wild type receptor, -MSH was twice as potent as ACTH. Chimera 4C3 is the next most potent recep tor. It was 10-fold less potent than the wild type receptor, and -MSH was twice as potent as ACTH. The only hMC2R chimera that exhibited any activity was 2C5. This receptor was significantly less potent than the wild type receptor, but -MSH was still a full agonist. ACTH reach ed 100% stimulation as compared to the forskolin control, but wa s a micromolar agonist inst ead of a nanomolar agonist. Table 7-4. Functional activity of -MSH and ACTH(1-24) at selected chimeras. Cell Line EC50 (nM) Ligand hMC4R 4C1 4C3 2C5 -MSH 4.980.1 326 5910 276301260 ACTH(1-24) 11.52.7 6611 1138 6306840 EC50 values calculated from the average of at least three experiments. Functional Assay: cAMP As the luciferase assay did not prove eff ective for assaying the chimeric receptors expressed in OS3 cells, the cAMP assay was us ed. However, once again no significant response was observed in the OS3 cells generated in this study. It is unknown why the cAMP assay failed as it was used earlier in this st udy to characterize the fi rst Flag-hMC2R constr uct in OS3 cells. It may be that the receptor expression was too low to produce a significant re sponse. As a result, no functional activity was obtained for OS3 cells.

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221 Discussion Chimeras in HEK293 Cell Lines The HEK293 cell line is derived from a culture of human embryonic kidney cells transformed with sheared DNA fr om the adenovirus 5 virus.224 It is a valuable cell line often used for protein expression and functional recep tor assays. The cell line does not contain any endogenous melanocortin receptors, but will express functional MC1,3-5 receptors upon transfection. The MC2R, however is not functional when ex pressed in this cell line.84,85 The hMC2R is only functional in cell lines of adre nal origin such as the Y1, Y6, and OS3 cell lines.228,238 This study seeks to determine the reas ons why the hMC2R is not functional in HEK293 cells and why the hMC2R is stimulat ed by ACTH but not by the other endogenous melanocortin ligands. The first area of interest explored was the possibility of an accessory protein which may be expressed selectively in adrena l cells and is necessary for MC2R activity. Two classes of accessory proteins were investigated, the RA MPs and MRAP. The RAMP family has been shown to associate with calcitonin receptor fam ily proteins and is essential for its activity. MRAP has recently been shown to be involve d in MC2R expression and functional activity.86 During the course of this dissert ation work, it was reported that co-transfection of MRAP with the MC2R into HEK293 cells re sulted in a functional receptor.362 In this study, RT-PCR was used to determine the gene expression of RA MP1-3 and MRAP in HEK293 transfected with the hMC4R gene and OS3 cell lines tr ansfected with the hMC2R gene One group of cells of each type was stimulated with 10-7M ACTH(1-24) for 6 h prior to mR NA isolation. Results are shown in Figure 7-6. The only accessory protein mRNA to be detected were RAMP 2 and 3 in unstimulated hMC4R-HEK cells. Upon ACTH stimul ation of this cell type, RAMP2 expression levels were significantly inhi bited and RAMP3 was no longer detected. No RAMPs were found

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222 in the OS3 cells. These data indicate that the RAMP family of protei ns is most likely not involved in hMC2R activity. MRAP mRNA was not detected in any cell types. This may be misleading, however, as the human MRAP gene pr obe was used in the experiments and the OS3 cell line is a mouse cell line (s ee Figure 7-7 for gene sequence alignment). Though the related Y1 and Y6 lines have been shown to express MRAP,86 the OS3 cell line has not been tested in the literature. It is likely that MRAP would be present in the OS3 cells if a mouse probe were used. These data represent, to the best of the au thor’s knowledge, the firs t examples of RAMP expression in HEK and OS3 cell lines. Following the RT-PCR experiments, a chimeric receptor approach was applied to the problem of why the hMC2R is not expresse d in HEK cells and why the hMC2R is only stimulated by ACTH. This method involves the substitution of large segments of one receptor with another receptor. This is done to investigat e the effects of large groups of amino acids on receptor structure and function. Since the hMC4R is well-characterized, it was used along with the mMC2R to generate chimeric receptors. Figu re 7-3 shows the chimeras generated in this study. A recent paper by Chen et al. describes the analysis of hMC4R chimeras created by the substitution of TM regions from the hMC2R in order to investigate th e regions of the hMC4R necessary for -MSH binding.369 It was determined that TM2, TM3 and TM6 of the hMC4R were involved in -MSH binding.369 The chimeras generated here represent larger regions of the receptors, including intracellular and extra cellular loops and both the hMC4R and its corresponding hMC2R chimera were made to serve as internal controls. A summary of the FACS data is presented in Figure 7-11. Expression data and mean fluorescence are presented both as raw data and as data normalized to the wild type. Expression data gives information about where the receptor is localized within the cell. The chimera data can

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223 be normalized to the wild type expression to show if the surface expression is increased or decreased. Surface expression is very important as the ligand cannot bind to the receptor if it is retained inside the cell. One detail noticed duri ng FACS analysis can be seen on the histogram analyses in Appendix C and Figure 7-13. It appears that there are two populations of receptors in the cells. One group has a higher ex pression or Flag-antibody binding le vel than the other. It may be that there are possibly two cell populations, one with higher expr ession, or it could be that one population has a greater affinity between the Flag and anti-Flag antibody than the other. Another possibility is that some receptors may be in a conformation that hides or covers up the Flag tag so that the antibody cannot access the tag as efficiently. FACS analysis revealed that chimeras 2C1, 2C 3 and 2C6 have a similar total expression as the hMC2R wild type, but surface expression is increased significantly. Amongst the hMC4R chimeras, all chimeras have a similar surface and total expression as the hMC4R wild type, though 4C4 has slightly less surface expression. Anot her parameter that may be analyzed from FACS data is the mean fluorescence (MF). This re veals the relative recept or expression per cell. A high MF value indicates that each cell is ex pressing a high concentration of receptor. A low MF value indicates that each cell expresses a low level of receptor. When each chimera is plotted as surface expression versus MF (Figure 7-16) it is revealed that that the 2C1, 2C3 and 2C6 receptors, which had an increased surface expressi on compared to the hMC2R wild type, also all express high levels of receptor. Interestingly, when the chimeras were tested for functional activity using the luciferase a ssay (Figure 7-14 and 7-15, Table 7-4), the 2C5 chimera was the only hMC2R chimera to have functional activit y. The hMC2R chimeras expressing high levels of receptor were not functional. This leads to the idea that overe xpression of the receptor may be inhibiting activity. When extremely high levels of receptor are continually expressed, it becomes

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224 difficult for the cellular machinery to keep up. With high levels of expression, one or more signaling factors may be titrated, leading to a loss of activity due to improper folding or membrane insertion. A positive forskolin response during stimulation indicates that the signal transduction machinery is probably still functional. Another theory is that with so much surface expression, there may simply be too many receptors in a small area, leading to overcrowding and improper receptor conformation. Chimera 2C5, which is the only active hMC2R chimera, expresses much lower levels of receptor than 2C1, 2C3 and 2C6, which are not active, lending a level of credence to this theory. Figure 7-16. Correlation plots of HEK hMC2R chimera data; % Surface Expression vs. Mean Fluorescence. Blue circle surrounds chimeras 2C1, 2C3, and 2C6. Red circle shows chimera 2C5. When the hMC4R receptors were tested for f unctional activity, only the wild type receptor, 4C1 and 4C3 displayed any activity. Additionally, among the chimeras with activity, there was no preference for one ligand over anothe r. It should be noted that the EC50 value for -MSH at the wild type hMC4R was over 10-fold higher than expected.42,43,56,370 This may be a result of transfection efficiency or other problems which may ha ve occurred during detection. Additionally, no difference in liga nd selectivity was observed betw een the wild type hMC4R and the active chimeras 4C1, 4C3 and 2C5. This experi ment did not answer the question about ligand selectivity. Functional assay of the chimeras in OS3 cells may have helped to clarify this

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225 question, but as no functional response was ab le to be obtained from the OS3 cells, no conclusions about ligand selectiv ity in the hMC2R may be drawn. N-Terminus (EC) hMC1R MSSVVEFTMAVQGSQRRLLGSLNSTPTAIPQLGLAANQTGARCLEVSIS hMC2R MKHIINSYENINNTARNNSDCPRVVLP hMC3R MSSVVEFTMSIQKTYLEGDFVFPVSSSSFLRTLLEPQLGSALLTAMNASCCLPSVQPTLPNGSEHLQAPFFSNQSSSAFCEQVFIK hMC4R MVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS hMC5R MSSFHLHFLDLNLNATEGNLSGPNVKNKSSPCEDMGIA 2C1 MVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS 2C2 MKHIINSYENINNTARNNSDCPRVVLP 2C3 MKHIINSYENINNTARNNSDCPRVVLP 2C4 MKHIINSYENINNTARNNSDCPRVVLP 2C5 MKHIINSYENINNTARNNSDCPRVVLP 2C6 MKVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS 4C1 MKHIINSYENINNTARNNSDCPRVVLP 4C2 MKVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS 4C3 MKVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS 4C4 MKVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS 4C5 MKVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVS 4C6 MKHIINSYENINNTARNNSDCPRVVLP TM1 Loop 1 (IC) hMC1R DGLFLSLGLVSLVENALVVATIAK NRNLHS hMC2R EEIFFTISIVGVLENLIVLLAVFK NKNLQA hMC3R PEVFLSLGIVSLLENILVILAVVR NGNLHS hMC4R PEVFVTLGVISLLENILVIVAIAK NKNLHS hMC5R VEVFLTLGVISLLENILVIGAIVK NKNLHS 2C1 PEVFVTLGVISLLENILVIVAIAK NKNLHS 2C2 EEIFFTISIVGVLENLIVLLAVFK NKNLQA 2C3 EEIFFTISIVGVLENLIVLLAVFK NKNLQA 2C4 EEIFFTISIVGVLENLIVLLAVFK NKNLQA 2C5 EEIFFTISIVGVLENLIVLLAVFK NKNLQA 2C6 PEVFVTLGVISLLENILVIVAIAK NKNLHS 4C1 EEIFFTISIVGVLENLIVLLAVFK NKNLQA 4C2 PEVFVTLGVISLLENILVIVAIAK NKNLHS 4C3 PEVFVTLGVISLLENILVIVAIAK NKNLHS 4C4 PEVFVTLGVISLLENILVIVAIAK NKNLHS 4C5 PEVFVTLGVISLLENILVIVAIAK NKNLHS 4C6 EEIFFTISIVGVLENLIVLLAVFK NKNLQA TM2 Loop 2 (EC) hMC1R PMYCFICCLALSDLLVSGSNVLETAVILL LEAGALVARAAVLQQL hMC2R PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA hMC3R PMYFFLCSLAVADMLVSVSNALETIMIAIVHSDY LT FED QFIQHM hMC4R PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI hMC5R PMYFFVCSLAVADMLVSMSSAWETITIYL LNNKHLVIADAFVRHI 2C1 PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA 2C2 PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI 2C3 PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA 2C4 PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA 2C5 PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI 2C6 PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI 4C1 PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI 4C2 PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA 4C3 PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI 4C4 PMYFFICSLAVADMLVSVSNGSETIVITL LN STDTDAQSFTVNI 4C5 PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA 4C6 PMYFFICSLAISDMLGSLYKILENILIIL RNMGYLKPRGSFETTA TM3 Loop 3(IC)

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226 hMC1R DNVIDVITCSSMLSSLCFLGAIAVDRYISI FYALRYHSIVT hMC2R DDIIDSLFVLSLLGSIFSLSVIAADRYITI FHALRYHSIVT hMC3R DNIFDSMICISLVASICNLLAIAVDRYVTI FYALRYHSIMT hMC4R DNVIDSVICSSLLASICSLLSIAVDRYFTI FYALQYHNIMT hMC5R DNVFDSMICISVVASMCSLLAIAVDRYVTI FYALRYHHIMT 2C1 DDIIDSLFVLSLLGSIFSLSVIAADRYITI FHALRYHSIVT 2C2 DNVIDSVICSSLLASICSLLSIAVDRYITI FHALRYHSIVT 2C3 DDIIDSLFVLSLLGSIFSLSVIAADRYFTI FYALQYHNIMT 2C4 DDIIDSLFVLSLLGSIFSLSVIAADRYITI FHALRYHSIVT 2C5 DNVIDSVICSSLLASICSLLSIAVDRYFTI FYALQYHNIMT 2C6 DNVIDSVICSSLLASICSLLSIAVDRYITI FHALRYHSIVT 4C1 DNXIDSVICSSLLASICSLLSIAVDRYFTI FYALQYHNIMT 4C2 DDIIDSLFVLSLLGSIFSLSVIAADRYFTI FYALQYHNIMT 4C3 DNVIDSVICSSLLASICSLLSIAVDRYITI FHALRYHSIVT 4C4 DNVIDSVICSSLLASICSLLSIAVDRYFTI FYALQYHNIMT 4C5 DDIIDSLFVLSLLGSIFSLSVIAADRYITI FHALRYHSIVT 4C6 DDIIDSLFVLSLLGSIFSLSVIAADRYFTI FYALQYHNIMT TM4 Loop 4 (EC) hMC1R LPRARRAVAAIWVASVVFSTLFI AYYDH hMC2R MRRTVVVLTVIWTFCTGTGITMV IFSHH hMC3R VRKALTLIVAIWVCCGVCG V V FIVYS hMC4R VKRVGIIISCIWAACTVSGILFI IYSDS hMC5R ARRSGAIIAGIWAFCTGCGIVFI LYSES 2C1 MRRTVVVLTVIWTFCTGTGITMV IFSHH 2C2 MRRTVVVLTVIWTFCTGTGITMV IFSHH 2C3 VKRVGIIISCIWAACTVSGILFI IYSDS 2C4 MRRTVVVLTVIWTFCTGTGITMV IFSHH 2C5 VKRVGIIISCIWAACTVSGILFI IYSDS 2C6 MRRTVVVLTVIWTFCTGTGITMV IFSHH 4C1 VKRVGIIISCIWAACTVSGILFI IYSDS 4C2 VKRVGIIISCIWAACTVSGILFI IYSDS 4C3 MRRTVVVLTVIWTFCTGTGITMV IFSHH 4C4 VKRVGIIISCIWAACTVSGILFI IYSDS 4C5 MRRTVVVLTVIWTFCTGTGITMV IFSHH 4C6 VKRVGIIISCIWAACTVSGILFI IYSDS TM5 Loop 5 (IC) hMC1R VA VLLCLVVFFLAMLVLMAVLYVHMLARACQHAQG IARLHKRQRPVHRPVHQ hMC2R VPT VITFTSL FPLMLVFILCLYVHMFLLARSHTRK ISTLP hMC3R ESKMVIVCLITMFFAMMLLMGTLYVHMFLFARLHVKR IAALPPADGVAPQQH hMC4R SA VIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ hMC5R TY VILCLISMFFAMLFLLVSLYIHMFLLARTHVKR IAALPGASSARQRTSMQ 2C1 VPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRK ISTLP 2C2 VPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRK ISTLP 2C3 SAVIICLITMFFTMLALMASLYVHMFLLARSHTRK ISTLP 2C4 VPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRK ISTLP 2C5 SAVIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ 2C6 VPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRK ISTLP 4C1 SAVIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ 4C2 SAVIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ 4C3 VPTVITFTSLFPLMLVFILCLYVHMFLMARLHIKR IAVLPGTGAIRQ 4C4 SAVIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ 4C5 VPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRK ISTLP 4C6 SAVIICLITMFFTMLALMASLYVHMFLMARLHIKR IAVLPGTGAIRQ TM6 Loop 6(EC) hMC1R GFGLKGAVTLTILLGIFFLCWGPFFLHLTLIVL CPEHPTCGC hMC2R RANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC hMC3R SCMKGAVTITILLGVFIFCWAPFFLHLVLIIT CPTNPYCIC hMC4R GANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC hMC5R GAVT VTMLLGVFTVCWAPFFLHLTLMLS CPQNLYCSR 2C1 RANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC

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227 2C2 RANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC 2C3 RANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC 2C4 RANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC 2C5 GANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC 2C6 RANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC 4C1 GANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC 4C2 GANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC 4C3 GANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC 4C4 GANMKGAITLTILLGVFIFCWAPFVLHVLLMTF CPSNPYCAC 4C5 RANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC 4C6 GANMKGAITLTILIGVFVVCWAPFFLHLIFYIS CPQNPYCVC TM7 C-terminus (IC) hMC1R IFKNFNLFLALIICNAIIDPLIYAFHSQ ELRRTLKEVLTCSW hMC2R YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW hMC3R YTAHFNTYLVLIMCNSVIDPLIYAFRSL ELRNTFREILCGCNGMNLG hMC4R FMSHFNLYLILIMCNDIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY hMC5R FMSHFNMYLILIMCNSVMDPLIYAFRSQ EMRKTFKEIICCRGFRIACSFPRRD 2C1 YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW 2C2 YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW 2C3 YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW 2C4 FMSHFNLYLILIMCNSIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY 2C5 YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW 2C6 YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW 4C1 FMSHFNLYLILIMCNSIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY 4C2 FMSHFNLYLILIMCNSIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY 4C3 FMSHFNLYLILIMCNSIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY 4C4 YMSLFQVNGMLIMCNAVIDPFIYAFRSP ELRDAFKKMIFCSRYW 4C5 FMSHFNLYLILIMCNSIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY 4C6 FMSHFNLYLILIMCNSIIDPLIYALRSQ ELRKTFKEIICCYPLGGLCDLSSRY Figure 7-17. Amino acid alignment of all me lanocortin receptors and chimeras. IC = intracellular, EC = extrace llular, IL = intracellular loop, EL = extracellular loop. Significant differences between the hM C2R and hMC4R and chimeras with functional activity are highlighted. Blank spaces inserted for alignment purposes. Based on preliminary FACS and functional data, the roles of each receptor were not clearly seen. Therefore all melanocortin receptors and chim eras were aligned with divisions between the proposed loop and TM regions based on previo us melanocortin alignments (Figure 7-17).43,49 These amino acid alignments revealed interestin g information about the structural differences between the hMC2R and the other melanocortin receptors. When looking at the sequences, the first major difference is seen in EL2. The hMC1, 3-5R all contain significa nt sequence similarity, however, the hMC2R differs greatly from the othe r receptors. Additionally, IL5 is significantly different in the other melanocortin receptors than in the hMC2R. As all wild type receptors and chimeras that express the combination of EL2 and IL5 similar to the hMC4R are active in HEK

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228 cells (4C1, 4C3, 2C5), and the hMC2R and chimeras that contain either the EL2 or IL5 from hMC2R are not active in HEK cells, it is possible th at these loops are nece ssary for activity in HEK cells. A protein database search of the IL5 of the MC1,3-5R revealed that there is some sequence similarity between this loop and othe r GPCRs. Among the classe s of receptors that contain homologous sequences are the olf actory receptors of several species371-375 and taste receptors, which have been shown to be active in HEK cells,376 among many others. In order to prove whether or not these loops are essential for activity in HE K cells, hMC2R chimeras should be made which incorporate the EL2 and/or IL5 from the hMC4R into the hMC2R template. Functional assay of these new chimeras shoul d show whether activity in conferred on the hMC2R with the introduction of one or both of these loops. Chimeras in OS3 Cell Lines Along with the HEK293 cell line, all chimeras were stably transfected into OS3 cells, which is an adrenal cell line developed from th e Y1 adrenocortical tumo r cell line. These cell lines contain a wild type MC 2R gene, however this DNA sequ ence is not functional due to growth in the presence of forskolin.228 The cells do, however, cont ain an intact cAMP signal transduction pathway, resulting in a functional transfected MC2R.228,229 FACS analysis of OS3 wild type receptors and chimeras resulted in similar expression patterns as was seen in HEK cells. The main difference is that expression levels were significantly lower in OS3 cells. Figure 7-12 summarizes the FACS data showing percent expression and mean fluorescence levels. When co mpared to wild type receptors, chimera 2C1, 2C2, and 2C3 showed increased surface expre ssion and 2C5 expression was similar to the hMC2R expression. Chimeras 4C2 and 4C5 exhib ited increased expression as compared to hMC4R wild type, though 4C4 showed significantly decreased expression levels. When MF data is compared to wild type, chimeras 2C1, 2C2, 2C 3, 2C5 and 2C6 all had decreased MF values as

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229 compared to the hMC2R, and chimera 4C4 show ed significantly decreased fluorescence levels compared to the hMC4R, but the other hMC4R chimeras had similar expression as compared to wild type. Figure 7-18. Time course of HEK and OS3 cell lines transiently transfected with GFP and incubated at 35oC and 3% CO2. HEK cells were transf ected using the calcium phosphate method. OS3 cells were tran sfected using Fugene 6 (Roche). It has been reported that the luciferase a ssay may be used with both HEK and OS3 cell types.238 However, after extensive testing, no lucifera se activity was observed in OS3 cells either with agonist stimulation or with forskolin st imulation, which increase s cAMP levels without receptor involvement. Furthermore, the cAMP assa y, which has been extensively used to test the hMC2R in OS3 cells, was not able to detect a functional response in th ese studies. It is unknown why the assays did not work in this case. One is sue is possibly transfection efficiency. OS3 cells are notoriously difficult to transfect (<2% tran sfection efficiency), and these are no different. Figure 7-12 shows FACS analysis data from the OS3 chimeras. Compared with HEK293 chimeras (Figure 7-11) receptor levels were si gnificantly lower in the OS3 cells. Figure 7-18

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230 shows both HEK and OS3 cells transfected with a GFP plasmid (experiment s performed with the help of Dr. Hendrik Luesch). Previous studies had shown that the calcium phosphate method is ineffective using OS3 cells (data not shown), so the lipid transfection r eagent Fugene 6 (Roche) was used to transfect the OS3 cells. A time course is shown in Figure 718, and it is clear that HEK cells transfect very efficiently, but th e OS3 cell line does not take up the DNA very efficiently. It may be that the stable cell lines simply did not express e nough of the receptor to produce a functional response. In th e future, it may be possible to use a different cell line which may transfect better than OS3 cells to test function of the hMC2R chimeras. Conclusions and Future Directions In summary, this study has identified new receptor chimeras of the hMC2R and hMC4R which have activity when stim ulated with ACTH(1-24) and -MSH, though no ligand selectivity was observed. All chimeras were expressed on the surface of both HEK and OS3 cells, though OS3 expression was very low. Chimeras 4C1, 4C3 and 2C5 are the only chimeras to have functional activity in HEK cells when tested with the luciferase assay. These chimeras are unique in that they are the only chimeras made which co ntain both the EL3 and IL5 from the hMC4R. It is suggested that these domains may be important for activity in HEK cells. Future studies should include the construction of hMC2R chimeras whic h contain one or both of these loops from the hMC4R to test their importance for HEK activit y. Another important experiment that should be performed is binding studies of the chimeras w ith either iodinated ACTH or NDP-MSH. These studies would allow the evaluation of the ligan d-receptor interaction and determination of regions of the receptors required for li gand binding versus signal transduction. OS3 cells stably transfected with the chimeras were shown to express the receptor on the cell surface, however expression levels were very low and no functional activity was observed. It may be possible to use a different cell line which is able to express a functional hMC2R to test

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231 these chimeras for activity. Binding studies w ould also be extremely valuable in the characterization of these chimeras. Gene expression assays performed on HEK and OS3 cells represent the first studies into the expression of RAMP1-3 and MRAP in these cell lines. No RAMPs were observed in the OS3 cells, though RAMP2 and 3 were present in unstimulated HEK cells but expression was significantly decreased when the cells were stimulated with ACTH(1-24). No MRAP was observed in any cell lines, but the human probe wa s used on the murine OS3 cell line so it is unknown whether mouse Mrap is present in this ce ll line. Future studies of hMC2R expression in HEK cells should include co-transfection of MR AP as it has recently been shown to confer activity to the hMC2R in HEK cells.362 These studies represent importa nt advancements in the characterization of the hMC2R. Novel chimeras were created that have activity in HEK293 cells, which are not able to functionally express the wild type receptor. Furt her studies are required to fully elucidate the functional and binding activity of these chimeras. It is hoped that these discoveries may someday aid in the understanding of the role of the hMC2R in stress and adrenal disorders.

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232 CHAPTER 8 CONCLUDING REMARKS Structure-Activity Relationship Studies Of Peptides: A Study Of Intramolecular CationInteractions In Melanocortin Agonists A nd The Effects On Agonist And Antagonist Selectivity In this study, MTII, a cyclic synthetic melanocortin peptide, was modified at the arginine and D-phenylalanine positions to investigate the role cationinteractions may play in the agonist or antagonist potency of this ligand at the melanocortin receptors. It was hypothesized that when combined with any one of six aromatic systems [Phe, DPhe, Nal(1’), DNal(1’), Nal(2’), or DNal(2’)], peptides with an arginine should exhibi t the greatest potency and those with an alanine should have a si gnificant loss of activ ity, with lysine peptides exhibiting an intermediate potency. Additiona lly, in accordance with the melanocortin antagonist SHU9119, compounds containing a DNal(2’) should exhibit an tagonist activity. Napthyl alanine derivatives were used because the double aromatic ring result s in a greater negative electrostatic potential than the single ring of phenylalanine, and thus should interact more strongly with the cation. Several conclusions may be drawn from the information obtained in this study. First it appears that the original hypothesis is corre ct and there may be a significant cationinteraction between the Arg and the adjacent aromatic am ino acid in melanocortin ligands. This was confirmed by the observation that in the majority of peptides synthesized in this study the Arg compound was the most potent, followed by Lys, and finally Ala as the least potent compound. It was also observed that in each aromatic system tested, the D form of the amino acid was more potent than the L form. Additionally, DNal(2’) is the only aromatic system tested which has antagonist activity at the mM C3R and mMC4R. An interest ing observation is that the Nal(1’)/Ala combination results in a peptide with antagonist activity at the mMC4R. These

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233 studies are significant as they pr ovide important information which may be used in the future to design new peptide and non-peptide liga nds for the melanocortin receptors. Design Of Peptidomimetics And Small Molecu les: 1,4-Benzodiazepine-2,5-Diones As nM Melanocortin Agonists The benzodiazepine template has been widely st udied as a scaffold for non-peptide drugs. As many targets are members of the G-prot ein coupled receptor super family (i.e. cholecystokinin, -opioid, oxytocin, and endothelin recep tors), it was hypothesized that benzodiazepine derivatives could be designed as melanocortin ligands. The 1,4-benzodiazepine-2,5-dione library synt hesized in this study was designed to interact with proposed binding sites in th e melanocortin receptors. Homology molecular modeling of the mMC1R44,341 and the mMC4R43,48-50,342 suggest the presence of hydrophobic and electrostatic binding pockets whic h are essential for binding of the melanocortin ligands to the receptor. Based on the results of this study, it was proposed that the p(Cl)-phenyl ring of the benzodiazepine and an aryl side chain may in teract with the hydrophobi c pockets and that a cationic component may interact with the anionic binding pocket. The benzodiazepines reported herein were de signed based on a set of 1,4-benzodiazepine2,5-dione compounds synthesized by Christine G. Joseph of the Haskell-Luevano laboratory that were shown by functional analysis to have activity at the melanocortin receptors. Based on these interesting pha rmacology results, a new series of compounds (Compounds 39-58) were designed in order to investigate the structural requirements necessary for benzodiazepine activity at the melanocortin re ceptors, and to probe the structure-activity relationships at each point of diversity. Future studies of this class of compounds should focus on the incorporation of aliphatic groups at R2, as well as halogens at R1. The R3 position seems to be less strict, allowing for the

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234 incorporation of building blocks mimicking amino acid side chains of Phe, Lys, Arg, and Trp as well as others. The His side chain cannot be inco rporated using this synthetic scheme due to the low stability of the imidazole side chain unde r the reductive environm ent necessary for its addition. It may also be interesting to incorporate substituted derivatives of Phe, as some of these have been shown to result in compounds w ith activity at the melanocortin receptors.7 In conclusion, this research has resulted in the identification of several benzodiazepine derivatives that exhibit agonist activity at the mouse melanocortin receptors. These compounds appear to have the most activity at the mMC1R, with only a few compounds exhibiting significant activity at the other receptors. These compounds are easy to synthesize due to the use of solid phase organic synthesis and may be r eadily purified allowing for the design of a large number of compounds. When substituted with build ing blocks that mimic the active sequence of peptide melanocortin ligands, this template has the potential to cr eate compounds with increasing potency that may be used in the future as drugs to treat obesity. Construction And Characteri zation Of Melanocortin-2 And -4 Receptor Chimeras For many years ligands have been screened at the melanocortin receptors using the galactosidase assay. This assay works well with receptors expressed in HEK293 cells and the pCRE/ -gal plasmid transiently transfected into the cells before stimulation.209 This method is effective for cells expressing the MC1R, MC3R, MC4R, or MC5R, however, when the MC2R is stably transfected into HEK 293 cells, th ere is no functional response in the -galactosidase assay.83 As has been previously stated, it is known that stimulation of OS3 cells stably transfected with the hMC2R gene results in a functional response. Convers ely, HEK293 cells that contain the hMC2R do not produce a functiona l response when stimulated.84,85 Amino acid alignment of the hMC2R with the hMC4R has identified ke y amino acid changes in the transmembrane

PAGE 235

235 regions which may be involved in ligand binding a nd/or signal transduction (Figure 7-1). To test the hypothesis that these amino acids are involv ed in the production of a functional hMC2 receptor in HEK293 cells, chimeric receptors were made to identify the role each transmembrane domain plays in ligand binding and signal tran sduction in the hMC2R. Entire transmembrane regions from the hMC4R were inserted into the hMC2R gene and these chimeric receptors stably transfected into OS3 cells and HEK293 cells. Foll owing construction of chimeras and generation of stable cell lines, the expre ssion and localization of the recep tor was determined using FACS (Fluorescence Activated Cell Sorting). Functional assay of all chimeras in HEK293 cells was also performed to measure the response of the receptor to agonist stimulation. In summary, this study has identified new receptor chimeras of the hMC2R and hMC4R which have activity when stim ulated with ACTH(1-24) and -MSH, though no ligand selectivity was observed. All chimeras were expressed on the surface of both HEK and OS3 cells, though OS3 expression was very low. Chimeras 4C1, 4C3 and 2C5 are the only chimeras to have functional activity in HEK cells when tested with the luciferase assay. These chimeras are unique in that they are the only chimeras made which co ntain both the EL3 and IL5 from the hMC4R. It is suggested that these domains may be important for activity in HEK cells. Future studies should include the construction of hMC2R chimeras whic h contain one or both of these loops from the hMC4R to test their importance for HEK activit y. Another important experiment that should be performed is binding studies of the chimeras w ith radioactively labele d ACTH or NDP-MSH. These studies would allow the ev aluation of the ligand-receptor in teraction and determination of regions of the receptors required for li gand binding versus signal transduction. OS3 cells stably transfected with the chimeras were shown to express the receptor on the cell surface, however expression levels were very low and no functional activity was observed. It

PAGE 236

236 may be possible to use a different cell line which is able to express a functional hMC2R to test these chimeras for activity. Binding studies w ould also be extremely valuable in the characterization of these chimeras. Gene expression assays performed on HEK and OS3 cells represent the first studies into the expression of RAMP1-3 and MRAP in these cell lines. No RAMPs were observed in the OS3 cells, though RAMP2 and 3 were present in unstimulated HEK cells but expression was significantly decreased when the cells were stimulated with ACTH(1-24). No MRAP was observed in any cell lines, but the human probe wa s used on the murine OS3 cell line so it is unknown whether mouse Mrap is present in this ce ll line. Future studies of hMC2R expression in HEK cells should include co-transfection of MR AP as it has recently been shown to confer activity to the hMC2R in HEK cells.362 These studies represent importa nt advancements in the characterization of the hMC2R. Novel chimeras were created that have activity in HEK293 cells, which are not able to functionally express the wild type receptor. Furt her studies are required to fully elucidate the functional and binding activity of these chimeras. It is hoped that these discoveries may someday aid in the understanding of the role of the hMC2R in stress and adrenal disorders.

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237 APPENDIX A 1H-NMR OF 1,4-BENZODI AZEPINE-2,5-DIONES All compounds were dissolved in DMSOd6 and spectra were obtaine d at room temperature.

PAGE 238

238Figure A-1. Compound 39 (KRW9-70E) 1H-NMR, 3-(4-aminobutyl)-1-benzyl-3,4-dihydro1H-benzo[e][1,4]diazepine-2,5-dione.

PAGE 239

239Figure A-2. Compound 40 (KRW5-37) 1H-NMR, 3-Benzyl-1-biphenyl-2-ylmethyl-3,4-dihyd ro-1H-benzo[e][1,4]diazepine-2,5-dione.

PAGE 240

240Figure A-3. Compound 41 (KRW 9-70J) 1H-NMR, 1-(3-(1-(biphenyl-2-ylmethyl)-2,5-dioxo-2,3,4,5-tetrah ydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea.

PAGE 241

241Figure A-4. Compound 42 (KRW9-70F) 1H-NMR, 3-(4-aminobutyl)-1-(biphenyl-2-ylmethyl)-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione.

PAGE 242

242Figure A-5. Compound 43 (KRW9-70A) 1H-NMR, 3-benzyl-1-(naphthalen-1-ylmethyl)-3,4-dihyd ro-1H-benzo[e][1,4]diazepine-2,5-dione.

PAGE 243

243Figure A-6. Compound 44 (KRW9-70K) 1H-NMR, 1-(3-(1-(naphthalen-1-ylmethyl)-2,5dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea.

PAGE 244

244Figure A-7. Compound 45 (KRW9-70G) 1H-NMR, 3-(4-aminobutyl)-1-(naphthalen-1-ylmethyl)-3,4-d ihydro-1H-benzo[e][1,4]diazepine-2,5-dione.

PAGE 245

245Figure A-8. Compound 46 (KRW4-91) 1H-NMR, 3-Benzyl-1-propyl-3,4-dihydro-1H -benzo[e][1,4]diazepine-2,5-dione.

PAGE 246

246Figure A-9. Compound 47 (KRW5-25) 1H-NMR, 3-Benzyl-1-butyl-3,4-dihydro-1Hbenzo[e][1,4]diazepine-2,5-dione.

PAGE 247

247Figure A-10. Compound 48 (KRW9-70B) 1H-NMR, 1,3-dibenzyl-8-methyl-3,4-dihydro-1H -benzo[e][1,4]diazepine-2,5-dione.

PAGE 248

248Figure A-11. Compound 49 (KRW9-70L) 1H-NMR, 1-(3-(1-benzyl-9-methyl-2,5-dioxo-2,3,4,5-tetrah ydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea.

PAGE 249

249Figure A-12. Compound 50 (KRW9-70H) 1H-NMR, 3-(4-aminobutyl)-1-benzyl-9-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione.

PAGE 250

250Figure A-13. Compound 51 (KRW9-70C) 1H-NMR, 1-(3-(1-benzyl-8-chloro-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea.

PAGE 251

251Figure A-14. Compound 52 (KRW6-121A) 1H-NMR, N-[3-(1-Benzyl-8-chloro-2,5-dioxo-2,3,4,5-tetrahydro -1H-benzo[e][1,4]diazepin-3-yl)-propyl]-guanidine.

PAGE 252

252Figure A-15. Compound 53 (KRW9-70D) 1H-NMR, 3-benzyl-1-(biphenyl-2-ylmethy l)-8-chloro-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione

PAGE 253

253Figure A-16. Compound 54 (KRW9-70M) 1H-NMR, 1-(3-(1-(biphenyl-2-ylmethyl)-8-chloro-2,5-dioxo-2,3,4,5 -tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea

PAGE 254

254Figure A-17. Compound 55 (KRW9-70I) 1H-NMR, 3-(4-aminobutyl)-1-(biphenyl-2-ylmethyl)-8-chloro3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione

PAGE 255

255Figure A-18. Compound 56 (KRW5-49) 1H-NMR, 3-Benzyl-8-chloro-1-naphthalen-2-ylmethyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione

PAGE 256

256Figure A-19. Compound 57 (KRW9-70N) 1H-NMR, 1-(3-(8-chloro-1-(naphthalen-1-ylmethyl)-2,5-dioxo-2,3,4 ,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propyl)urea

PAGE 257

257Figure A-20. Compound 58 (KRW7-05A) 1H-NMR, 3-(4-Amino-butyl)-8-chloro-1-naphthalen-2-ylmethyl -3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione

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258 APPENDIX B DNA SEQUENCES AND RESTRICTION MAPS OF CHIMERAS Red text indicates sequences inse rted from the opposite receptor.

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259 Figure B-1. Flag-hMC2R DNA Sequence Complete Sequence of Fl ag-hMC2R in pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TA TTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGG GC AGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTG GC TCCATCTTCAGCCTGTCTGTGATTGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCA CG GGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGAT CC CACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACACTGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCT GC CCAAGTAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTCA AA AAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Msc1 BseY1 Bgl2 BpuE1 | | | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAACCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTATAACTCT orf 1 > L Q A P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I L R Pml1 Eco57 | | AACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGAAGTCGG orf 1 > N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F S L Kpn1 Rsr2 Bae1a Bts1 Acc65 | Bae1b Ale1 Bsg1 | | | | | | | | TGTCTGTGATTGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCAT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 ACAGACACTAACGACGCCTGGCGATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATGCCAGTA orf 1 > S V I A A D R Y I T I F H A L R Y H S I V T M R R T V V V L T V I Ale1 Nco1 | BmgB1 Bbs1 | | | | CTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GACCTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTACGACCAG orf 1 > W T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M L V BspLU ApaL1 | Bgl2 SpAcc | | | | TTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACAC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 AAGTAGGACACGGAGATACACGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGTAGTGTG orf 1 > F I L C L Y V H M F L L A R S H T R K I S T L P R A N M K G A I T L Apa1 PspOM | Bbs1 BseY1 | | | | | | TGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCCTGCTA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 ACTGGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGGTTCATTGGGGATGACGCGGACGAT orf 1 > T I L L G V F I F C W A P F V L H V L L M T F C P S N P Y C A C Y SpDon SpAcc Sac1 BspLU Ear1 | BsrD1 BspE1 Bpu10| Bsm1 | | | | | || | CATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTACAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCCTCGGGTCTCGAGTCCCTGCGTAAG orf 1 > M S L F Q V N G M L I M C N A V I D P F I Y A F R S P E L R D A F BspM1 SpAcc BfuA1 Pst1| AlwN1 Xho1 Xba1 | || | | | AAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 901 ---------+---------+---------+---------+----945 TTTTTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > K K M I F C S R Y W

PAGE 260

260 Figure B-2. Chimera 2C1 DNA Sequence Complete sequence of Flag-hMC2R/2C1/pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CT CTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATC AG CTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTAGC CATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAA TATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCCTGTCTGTG AT TGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTG AT CTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACC CT CCCCAGAGCCAACATGAAAGGGGCCATCACACTGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCC TG CTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTCAAAAAGATGATCTTCTGCAGCAGG TA CTGGTAGCTCGAGTCTAGA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Blp1 Bsm1 Pst1 | BseY1 Bgl2 | | | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTAGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAATCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTAT orf 1 > K N L H S P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I BpuE1 Pml1 Eco57 | | | TTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AACTCTTTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGA orf 1 > L R N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F Kpn1 Rsr2 Bae1a Bts1 Acc65 | Bae1b Ale1 | | | | | | | TCAGCCTGTCTGTGATTGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTAC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 AGTCGGACAGACACTAACGACGCCTGGCGATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATG orf 1 > S L S V I A A D R Y I T I F H A L R Y H S I V T M R R T V V V L T Ale1 Bsg1 Nco1 | BmgB1 Bbs1 | | | | | GGTCATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CCAGTAGACCTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTAC orf 1 > V I W T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M BspLU ApaL1 | Bgl2 SpAcc | | | | CTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACCAGAAGTAGGACACGGAGATACACGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGT orf 1 > L V F I L C L Y V H M F L L A R S H T R K I S T L P R A N M K G A I Apa1 PspOM | Bbs1 BseY1 | | | | | | TCACACTGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGTGTGACTGGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGGTTCATTGGGGATGACGCG orf 1 > T L T I L L G V F I F C W A P F V L H V L L M T F C P S N P Y C A SpDon SpAcc Sac1 BspLU Ear1 | BsrD1 BspE1 Bpu10| | | | | | || CTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGAC 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 GACGATGTACAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCCTCGGGTCTCGAGTCCCTG orf 1 > C Y M S L F Q V N G M L I M C N A V I D P F I Y A F R S P E L R D BspM1 SpAcc Bsm1 BfuA1 Pst1| AlwN1 Xho1 Xba1 | | || | | | GCATTCAAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 1001 ---------+---------+---------+---------+---------+1051 CGTAAGTTTTTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > A F K K M I F C S R Y W

PAGE 261

261 Figure B-3. Chimera 2C2 DNA Sequence Complete sequence of Flag-hMC2R/2C2/pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TA TTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTA GCTTAGCTGTGGCTGATATGCTGGTG AGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCAT CC ATTTGCAGCCTGCTTTCAATTGCAGTGGACCGGT ACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCACGGGG ACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCC AC ACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACACTGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCC CA AGTAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTCAAAA AG ATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Blp1 SpDon | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTAGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAATCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAAT orf 1 > L Q A P M Y F F I C S L A V A D M L V S V S N G S E T I V I T L L T7Ter Ssp1 | | AACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACG orf 1 > N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S L L Bts1 Kpn1 Mfe1 Age1 Bae1a Bts1 Acc65 | Bae1b Ale1 Bsg1 | | | | | | | | | TTTCAATTGCAGTGGACCGGTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AAAGTTAACGTCACCTGGCCATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATGCCAGTAGAC orf 1 > S I A V D R Y I T I F H A L R Y H S I V T M R R T V V V L T V I W Ale1 Nco1 | BmgB1 Bbs1 | | | | GACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTACGACCAGAAG orf 1 > T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M L V F BspLU ApaL1 | Bgl2 SpAcc | | | | ATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACACTGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGGACACGGAGATACACGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGTAGTGTGACT orf 1 > I L C L Y V H M F L L A R S H T R K I S T L P R A N M K G A I T L T Apa1 PspOM | Bbs1 BseY1 | | BspLU | | | | | CCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCCTGCTACAT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGGTTCATTGGGGATGACGCGGACGATGTA orf 1 > I L L G V F I F C W A P F V L H V L L M T F C P S N P Y C A C Y M SpDon SpAcc Sac1 Ear1 | BsrD1 BspE1 Bpu10| Bsm1 | | | | || | GTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTCAAA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 CAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCCTCGGGTCTCGAGTCCCTGCGTAAGTTT orf 1 > S L F Q V N G M L I M C N A V I D P F I Y A F R S P E L R D A F K BspM1 SpAcc BfuA1 Pst1| AlwN1 Xho1 Xba1 | || | | | AAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 901 ---------+---------+---------+---------+-942 TTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > K M I F C S R Y W

PAGE 262

262 Figure B-4. Chimera 2C3 DNA Sequence Complete sequence of Flag-hMC2R/2C3/pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TA TTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGG GC AGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTG GC TCCATCTTCAGCCTGTCTGTGATTGCTGCGG ACCGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACG GTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTACA CATGTTCCTGCTGGCTCGATCC CACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACACTGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCT GC CCAAGTAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTCA AA AAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Msc1 BseY1 Bgl2 BpuE1 | | | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAACCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTATAACTCT orf 1 > L Q A P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I L R Pml1 Eco57 | | AACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGAAGTCGG orf 1 > N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F S L Age1 SpDon Rsr2 | Bpm1| | | || TGTCTGTGATTGCTGCGGACCGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTAT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 ACAGACACTAACGACGCCTGGCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATA orf 1 > S V I A A D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S C I SpDon | CTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGA orf 1 > W A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M L A BsrG1BspLU Bgl2 SpAcc | | | | CTCATGGCTTCTCTCTATGTACACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACAC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GAGTACCGAAGAGAGATACATGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGTAGTGTG orf 1 > L M A S L Y V H M F L L A R S H T R K I S T L P R A N M K G A I T L Apa1 PspOM | Bbs1 BseY1 | | | | | | TGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCCTGCTA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 ACTGGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGGTTCATTGGGGATGACGCGGACGAT orf 1 > T I L L G V F I F C W A P F V L H V L L M T F C P S N P Y C A C Y SpDon SpAcc Sac1 BspLU Ear1 | BsrD1 BspE1 Bpu10| Bsm1 | | | | | || | CATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTACAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCCTCGGGTCTCGAGTCCCTGCGTAAG orf 1 > M S L F Q V N G M L I M C N A V I D P F I Y A F R S P E L R D A F BspM1 SpAcc BfuA1 Pst1| AlwN1 Xho1 Xba1 | || | | | AAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 901 ---------+---------+---------+---------+----945 TTTTTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > K K M I F C S R Y W

PAGE 263

263 Figure B-5. Chimera 2C4 DNA Sequence Complete sequence of Flag-hMC2R/2C4/pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TA TTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGG GC AGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTG GC TCCATCTTCAGCCTGTCTGTGATTGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCA CG GGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGAT CC CACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCAC GTTAACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGT CCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCA AA GAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTC TA GA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Msc1 BseY1 Bgl2 BpuE1 | | | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAACCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTATAACTCT orf 1 > L Q A P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I L R Pml1 Eco57 | | AACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGAAGTCGG orf 1 > N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F S L Kpn1 Rsr2 Bae1a Bts1 Acc65 | Bae1b Ale1 Bsg1 | | | | | | | | TGTCTGTGATTGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCAT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 ACAGACACTAACGACGCCTGGCGATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATGCCAGTA orf 1 > S V I A A D R Y I T I F H A L R Y H S I V T M R R T V V V L T V I Ale1 Nco1 | BmgB1 Bbs1 | | | | CTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GACCTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTACGACCAG orf 1 > W T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M L V BspLU ApaL1 | Bgl2 SpAcc | | | | TTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACGT 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 AAGTAGGACACGGAGATACACGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGTAGTGCA orf 1 > F I L C L Y V H M F L L A R S H T R K I S T L P R A N M K G A I T L Apa1 PspOM | Hpa1 BseY1 | | Ssp1 | | | | | TAACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 ATTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAA orf 1 > T I L I G V F V V C W A P F F L H L I F Y I S C P Q N P Y C V C F SpDon BspE1 SpAcc Xmn1 | | | | CATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAG orf 1 > M S H F N L Y L I L I M C N S I I D P L I Y A L R S Q E L R K T F T7Ter Stu1 EcoR1 Pst1 | | | | AAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 TTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCGTGCGTTATATCTTCGGCTTAAGACGTC orf 1 > K E I I C C Y P L G G L C D L S S R Y Not1 Xho1 EcoRV BstX1 Eag1BsrB1| Xba1 | | | || | ATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1001 ---------+---------+---------+---1034 TATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 264

264 Figure B-6. Chimera 2C5 DNA Sequence Complete sequence of Flag-hMC2R/2C5/pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TA TTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTA GCTTAGCTGTGGCTGATATGCTGGTG AGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCAT CC ATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGG TT TCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTC AC ATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACGTTAAC CATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTC CTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAG AG CTCAGGGACGCATTCAAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Blp1 SpDon | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTAGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAATCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAAT orf 1 > L Q A P M Y F F I C S L A V A D M L V S V S N G S E T I V I T L L T7Ter Ssp1 | | AACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACG orf 1 > N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S L L SpDon Mfe1 Bts1 Bpm1| | | || TTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGAC orf 1 > S I A V D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S C I W SpDon | GGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAG orf 1 > A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M L A L BspLU SpDon Eci1 | | | ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTAT orf 1 > M A S L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G A N M Apa1 PspOM | Hpa1 Bbs1 BseY1 | | | | | | | TGAAGGGAGCGATTACGTTAACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 ACTTCCCTCGCTAATGCAATTGGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGGTTCATT orf 1 > K G A I T L T I L L G V F I F C W A P F V L H V L L M T F C P S N SpDon SpAcc BspLU Ear1 | BsrD1 BspE1 | | | | | CCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGGATGACGCGGACGATGTACAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCCTCGGGT orf 1 > P Y C A C Y M S L F Q V N G M L I M C N A V I D P F I Y A F R S P Sac1 BspM1 SpAcc Bpu10| Bsm1 BfuA1 Pst1| AlwN1 Xho1 Xba1 || | | || | | | GAGCTCAGGGACGCATTCAAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 901 ---------+---------+---------+---------+---------+---------+--963 CTCGAGTCCCTGCGTAAGTTTTTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > E L R D A F K K M I F C S R Y W

PAGE 265

265 Figure B-7. Chimera 2C6 DNA Sequence Complete sequence of Flag-hMC2R/2C5/pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CTCTGGAACCGC AGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGG AGAATATCTTAG TGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATT AAACAGTACAGA TACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACCGGT ACATCACCATCTTCCACGCACTGCGGTAC CACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGC TGTTCCCGCTGA TGCTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCACACTGACCATCCTGCTCGGGGT CTTCATCTTCTG CTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCC TTCATATATGCC TTCCGGAGCCCAGAGCTCAGGGACGCATTCAAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Bsm1 Pst1 SpDon | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGG orf 1 > K N L H S P M Y F F I C S L A V A D M L V S V S N G S E T I V I T T7Ter Ssp1 | | CTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGT orf 1 > L L N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S Bts1 Kpn1 Mfe1 Age1 Bae1a Bts1 Acc65 | Bae1b Ale1 Bsg1 | | | | | | | | | GCCTGCTTTCAATTGCAGTGGACCGGTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CGGACGAAAGTTAACGTCACCTGGCCATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATGCCA orf 1 > L L S I A V D R Y I T I F H A L R Y H S I V T M R R T V V V L T V Ale1 Nco1 | BmgB1 Bbs1 | | | | CATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTAGACCTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTACGAC orf 1 > I W T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M L BspLU ApaL1 | Bgl2 SpAcc | | | | GTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCATCA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CAGAAGTAGGACACGGAGATACACGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGTAGT orf 1 > V F I L C L Y V H M F L L A R S H T R K I S T L P R A N M K G A I T Apa1 PspOM | Bbs1 BseY1 | | | | | | CACTGACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAGTAACCCCTACTGCGCCTG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTGACTGGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGGTTCATTGGGGATGACGCGGAC orf 1 > L T I L L G V F I F C W A P F V L H V L L M T F C P S N P Y C A C SpDon SpAcc Sac1 BspLU Ear1 | BsrD1 BspE1 Bpu10| Bsm1 | | | | | || | CTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCA 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 GATGTACAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCCTCGGGTCTCGAGTCCCTGCGT orf 1 > Y M S L F Q V N G M L I M C N A V I D P F I Y A F R S P E L R D A BspM1 SpAcc BfuA1 Pst1| AlwN1 Xho1 Xba1 | || | | | TTCAAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 1001 ---------+---------+---------+---------+-------1048 AAGTTTTTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > F K K M I F C S R Y W

PAGE 266

266 Figure B-8. Flag-hMC4R DNA Sequence Complete Sequence of Fl ag-hMC4R in pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CT CTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATC AG CTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAA AC CATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATT GC AGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATT TA CTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTC CC CGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCT CA GAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAG AT CATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Bsm1 Pst1 SpDon | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGG orf 1 > K N L H S P M Y F F I C S L A V A D M L V S V S N G S E T I V I T T7Ter Ssp1 | | CTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGT orf 1 > L L N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S SpDon Mfe1 Bts1 Bpm1| | | || GCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTG 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAAC orf 1 > L L S I A V D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S C SpDon | TATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 ATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGAC orf 1 > I W A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M L BspLU SpDon Eci1 | | | GCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCAC orf 1 > A L M A S L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G A Apa1 PspOM | BseY1 | | Ssp1 | | | | CCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGTTATACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGG orf 1 > N M K G A I T L T I L I G V F V V C W A P F F L H L I F Y I S C P SpDon BspE1 | | TCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 AGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCC orf 1 > Q N P Y C V C F M S H F N L Y L I L I M C N S I I D P L I Y A L R SpAcc Xmn1 T7Ter Stu1 | | | | AGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACG 1001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1100 TCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCGTGC orf 1 > S Q E L R K T F K E I I C C Y P L G G L C D L S S R Y EcoRV Not1 Xho1 EcoR1 Pst1 | BstX1 Eag1BsrB1| Xba1 | | | | | || | CAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1101 ---------+---------+---------+---------+---------+-------1158 GTTATATCTTCGGCTTAAGACGTCTATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 267

267 Figure B-9. Chimera 4C1 DNA Sequence Complete sequence of Flag-hMC4R/4C1/pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TA TTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTAGC TGTGGCTGATATGCTGGTG AGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATNTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCAT CC ATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGG TT TCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTC AC ATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACT TA ATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAG AA CTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACA GT GGCGGCCGCTCGAGTCTAGA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Blp1 SpDon | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTAGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAATCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAAT orf 1 > L Q A P M Y F F I C S L A V A D M L V S V S N G S E T I V I T L L T7Ter Ssp1 | | AACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATNTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTANAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACG orf 1 > N S T D T D A Q S F T V N I D N X I D S V I C S S L L A S I C S L L SpDon Mfe1 Bts1 Bpm1| | | || TTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGAC orf 1 > S I A V D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S C I W SpDon | GGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAG orf 1 > A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M L A L BspLU SpDon Eci1 | | | ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTAT orf 1 > M A S L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G A N M Apa1 PspOM | BseY1 | | Ssp1 | | | | TGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 ACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTT orf 1 > K G A I T L T I L I G V F V V C W A P F F L H L I F Y I S C P Q N SpDon BspE1 | | TCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTT orf 1 > P Y C V C F M S H F N L Y L I L I M C N S I I D P L I Y A L R S Q SpAcc Xmn1 T7Ter Stu1 | | | | GAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATAT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 CTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCGTGCGTTATA orf 1 > E L R K T F K E I I C C Y P L G G L C D L S S R Y EcoRV Not1 Xho1 EcoR1 Pst1 | BstX1 Eag1BsrB1| Xba1 | | | | | || | AGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1001 ---------+---------+---------+---------+---------+-1052 TCTTCGGCTTAAGACGTCTATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 268

268 Figure B-10. Chimera 4C2 DNA Sequence Complete sequence of Flag-hMC4R/4C2/pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CT CTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATC AG CTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCA GCTTAGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAA TATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCCTGTCTGTG AT TGCTGCGGACCGGT ACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCAT TTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTC CT CCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGT CC TCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAA GA GATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTA GA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Blp1 Bsm1 Pst1 | BseY1 Bgl2 | | | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTAGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAATCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTAT orf 1 > K N L H S P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I BpuE1 Pml1 Eco57 | | | TTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AACTCTTTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGA orf 1 > L R N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F Age1 SpDon Rsr2 | Bpm1| | | || TCAGCCTGTCTGTGATTGCTGCGGACCGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAG 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 AGTCGGACAGACACTAACGACGCCTGGCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTC orf 1 > S L S V I A A D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S SpDon | TTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 AACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTAC orf 1 > C I W A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M BspLU SpDon Eci1 | | | CTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTC orf 1 > L A L M A S L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G Apa1 PspOM | BseY1 | | Ssp1 | | | | GTGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 CACGGTTATACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAAC orf 1 > A N M K G A I T L T I L I G V F V V C W A P F F L H L I F Y I S C SpDon BspE1 | | TCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTC 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 AGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAG orf 1 > P Q N P Y C V C F M S H F N L Y L I L I M C N S I I D P L I Y A L SpAcc Xmn1 T7Ter Stu1 | | | | CGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGC 1001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1100 GCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCG orf 1 > R S Q E L R K T F K E I I C C Y P L G G L C D L S S R Y EcoRV Not1 Xho1 EcoR1 Pst1 | BstX1 Eag1BsrB1| Xba1 | | | | | || | ACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1101 ---------+---------+---------+---------+---------+---------+1161 TGCGTTATATCTTCGGCTTAAGACGTCTATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 269

269 Figure B-11. Chimera 4C3 DNA Sequence Complete sequence of Flag-hMC4R/4C3/pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CT CTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATC AG CTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAA AC CATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATT GC AGTGG ACCGGTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTT CTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTCATCCTGTGCCTCTATGTACA CATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCC CGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCT CA GAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAG AT CATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Bsm1 Pst1 SpDon | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGG orf 1 > K N L H S P M Y F F I C S L A V A D M L V S V S N G S E T I V I T T7Ter Ssp1 | | CTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGT orf 1 > L L N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S Bts1 Kpn1 Mfe1 Age1 Bae1a Bts1 Acc65 | Bae1b Ale1 Bsg1 | | | | | | | | | GCCTGCTTTCAATTGCAGTGGACCGGTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CGGACGAAAGTTAACGTCACCTGGCCATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATGCCA orf 1 > L L S I A V D R Y I T I F H A L R Y H S I V T M R R T V V V L T V Ale1 Nco1 | BmgB1 Bbs1 | | | | CATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTAGACCTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTACGAC orf 1 > I W T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M L BsrG1BspLU SpDon Eci1 | | | | GTCTTCATCCTGTGCCTCTATGTACACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CAGAAGTAGGACACGGAGATACATGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCAC orf 1 > V F I L C L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G A Apa1 PspOM | BseY1 | | Ssp1 | | | | CCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGTTATACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGG orf 1 > N M K G A I T L T I L I G V F V V C W A P F F L H L I F Y I S C P SpDon BspE1 | | TCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 AGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCC orf 1 > Q N P Y C V C F M S H F N L Y L I L I M C N S I I D P L I Y A L R SpAcc Xmn1 T7Ter Stu1 | | | | AGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACG 1001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1100 TCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCGTGC orf 1 > S Q E L R K T F K E I I C C Y P L G G L C D L S S R Y EcoRV Not1 Xho1 EcoR1 Pst1 | BstX1 Eag1BsrB1| Xba1 | | | | | || | CAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1101 ---------+---------+---------+---------+---------+-------1158 GTTATATCTTCGGCTTAAGACGTCTATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 270

270 Figure B-12. Chimera 4C4 DNA Sequence Complete sequence of Flag-hMC4R/4C4/pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CT CTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATC AG CTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAA AC CATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATT GC AGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATT TA CTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTC CC CGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTAC GTTAACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCCAAG TAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGGAGCCCAGAGCTCAGGGACGCATTCAAAAAG AT GATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Bsm1 Pst1 SpDon | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGACCACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGG orf 1 > K N L H S P M Y F F I C S L A V A D M L V S V S N G S E T I V I T T7Ter Ssp1 | | CTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGT orf 1 > L L N S T D T D A Q S F T V N I D N V I D S V I C S S L L A S I C S SpDon Mfe1 Bts1 Bpm1| | | || GCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTG 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAAC orf 1 > L L S I A V D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S C SpDon | TATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 ATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGAC orf 1 > I W A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M L BspLU SpDon Eci1 | | | GCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCAC orf 1 > A L M A S L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G A Apa1 PspOM | Hpa1 Bbs1 BseY1 | | | | | | | CCAATATGAAGGGAGCGATTACGTTAACCATCCTGCTCGGGGTCTTCATCTTCTGCTGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTCTGCCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGTTATACTTCCCTCGCTAATGCAATTGGTAGGACGAGCCCCAGAAGTAGAAGACGACCCGGGGGAAACACGAAGTACAGGAGAACTACTGTAAGACGGG orf 1 > N M K G A I T L T I L L G V F I F C W A P F V L H V L L M T F C P SpDon SpAcc BspLU Ear1 | BsrD1 BspE1 | | | | | AAGTAACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAACGGCATGTTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCGG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 TTCATTGGGGATGACGCGGACGATGTACAGAGAGAAGGTCCACTTGCCGTACAACTAGTACACGTTACGGCAGTAACTGGGGAAGTATATACGGAAGGCC orf 1 > S N P Y C A C Y M S L F Q V N G M L I M C N A V I D P F I Y A F R Sac1 BspM1 SpAcc Bpu10| Bsm1 BfuA1 Pst1| AlwN1 Xho1 Xba1 || | | || | | | AGCCCAGAGCTCAGGGACGCATTCAAAAAGATGATCTTCTGCAGCAGGTACTGGTAGCTCGAGTCTAGA 1001 ---------+---------+---------+---------+---------+---------+--------1069 TCGGGTCTCGAGTCCCTGCGTAAGTTTTTCTACTAGAAGACGTCGTCCATGACCATCGAGCTCAGATCT orf 1 > S P E L R D A F K K M I F C S R Y W

PAGE 271

271 Figure B-13. Chimera 4C5 DNA Sequence Complete sequence of Flag-hMC4R/4C5/pcDNA3 AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCAC CT CTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATC AG CTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCA GCTTAGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAA TATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCCTGTCTGTG AT TGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTACGGTCATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTG AT CTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATGCTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACC CT CCCCAGAGCCAACATGAAAGGGGCCATCACGTTAAC CATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTG CTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCC CT GGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA Restriction Map Kpn1 BamH1 EcoR1 Hind3 Acc65 | Sac1 | Spe1 BstX1 | Nco1 SpAcc SpDon | | | | | | | | | | | AAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGGTGAACTCCACC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACCATGGCTCGAGCCTAGGTGATCAGGTCACACCACCTTAAGCCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGG orf 1 > M D Y K D D D D K V N S T Bsg1 Bsg1 BsrD1 | | | CACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTC orf 1 > H R G M H T S L H L W N R S S Y R L H S N A S E S L G K G Y S D G G Bsu36 | GGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTT orf 1 > C Y E Q L F V S P E V F V T L G V I S L L E N I L V I V A I A K N Blp1 Bsm1 Pst1 | BseY1 Bgl2 | | | | | CAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTAGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAATCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTAT orf 1 > K N L H S P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I BpuE1 Pml1 Eco57 | | | TTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AACTCTTTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGA orf 1 > L R N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F Kpn1 Rsr2 Bae1a Bts1 Acc65 | Bae1b Ale1 | | | | | | | TCAGCCTGTCTGTGATTGCTGCGGACCGCTACATCACCATCTTCCACGCACTGCGGTACCACAGCATCGTGACCATGCGCCGCACTGTGGTGGTGCTTAC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 AGTCGGACAGACACTAACGACGCCTGGCGATGTAGTGGTAGAAGGTGCGTGACGCCATGGTGTCGTAGCACTGGTACGCGGCGTGACACCACCACGAATG orf 1 > S L S V I A A D R Y I T I F H A L R Y H S I V T M R R T V V V L T Ale1 Bsg1 Nco1 | BmgB1 Bbs1 | | | | | GGTCATCTGGACGTTCTGCACGGGGACTGGCATCACCATGGTGATCTTCTCCCATCATGTGCCCACAGTGATCACCTTCACGTCGCTGTTCCCGCTGATG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CCAGTAGACCTGCAAGACGTGCCCCTGACCGTAGTGGTACCACTAGAAGAGGGTAGTACACGGGTGTCACTAGTGGAAGTGCAGCGACAAGGGCGACTAC orf 1 > V I W T F C T G T G I T M V I F S H H V P T V I T F T S L F P L M BspLU ApaL1 | Bgl2 SpAcc | | | | CTGGTCTTCATCCTGTGCCTCTATGTGCACATGTTCCTGCTGGCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAACATGAAAGGGGCCA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACCAGAAGTAGGACACGGAGATACACGTGTACAAGGACGACCGAGCTAGGGTGTGGTCCTTCTAGAGGTGGGAGGGGTCTCGGTTGTACTTTCCCCGGT orf 1 > L V F I L C L Y V H M F L L A R S H T R K I S T L P R A N M K G A I Apa1 PspOM | Hpa1 BseY1 | | Ssp1 | | | | | TCACGTTAACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGTGCAATTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACA orf 1 > T L T I L I G V F V V C W A P F F L H L I F Y I S C P Q N P Y C V SpDon BspE1 SpAcc | | | GTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAA 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 CACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTT orf 1 > C F M S H F N L Y L I L I M C N S I I D P L I Y A L R S Q E L R K Xmn1 T7Ter Stu1 EcoR1 | | | | ACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATT 1001 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1100 TGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCGTGCGTTATATCTTCGGCTTAA orf 1 > T F K E I I C C Y P L G G L C D L S S R Y EcoRV Not1 Xho1 Pst1 | BstX1 Eag1BsrB1| Xba1 | | | | || | CTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1101 ---------+---------+---------+---------+ 1140 GACGTCTATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 272

272 Figure B-14. Chimera 4C6 DNA Sequence Complete sequence of Flag-hMC4R/4C6/pcDNA3 AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGTGTGGTTTTGCCGGAGGAGA TATTTTTCACAA TTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGGGCAGCCTATA TAAGATCTTGGA AAATATCCTGATCATATTGAGAAACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCCTGTCT GTGATTGCTGCG GACCGGT ACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAG TAGTG CTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGC CATCCGCCAAGG TGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTC ATGTCTCACTTT AACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTG ACTTGTCTAGCA GATATTAAATGGGGACAGAGCACGCAATATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA Restriction Map Hind3 Nco1 SpAcc Xmn1 | | | | AAGCTTGCCGCCGCCATGGACTACAAGGACGACGACGACAAGAAGCACATTATCAACTCGTATGAAAACATCAACAACACAGCAAGAAATAATTCCGACT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTCGAACGGCGGCGGTACCTGATGTTCCTGCTGCTGCTGTTCTTCGTGTAATAGTTGAGCATACTTTTGTAGTTGTTGTGTCGTTCTTTATTAAGGCTGA orf 1 > M D Y K D D D D K K H I I N S Y E N I N N T A R N N S D C SpDon BssS1 BseR1| | || GTCCTCGTGTGGTTTTGCCGGAGGAGATATTTTTCACAATTTCCATTGTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAGGAGCACACCAAAACGGCCTCCTCTATAAAAAGTGTTAAAGGTAACAACCTCAAAACCTCTTAGACTAGCAGGACGACCGACACAAGTTCTTATTCTT orf 1 > P R V V L P E E I F F T I S I V G V L E N L I V L L A V F K N K N T7Ter Msc1 BseY1 Bgl2 BpuE1 | | | | | TCTCCAGGCACCCATGTACTTTTTCATCTGTAGCTTGGCCATATCTGATATGCTGGGCAGCCTATATAAGATCTTGGAAAATATCCTGATCATATTGAGA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGAGGTCCGTGGGTACATGAAAAAGTAGACATCGAACCGGTATAGACTATACGACCCGTCGGATATATTCTAGAACCTTTTATAGGACTAGTATAACTCT orf 1 > L Q A P M Y F F I C S L A I S D M L G S L Y K I L E N I L I I L R Pml1 Eco57 | | AACATGGGCTATCTCAAGCCACGTGGCAGTTTTGAAACCACAGCCGATGACATCATCGACTCCCTGTTTGTCCTCTCCCTGCTTGGCTCCATCTTCAGCC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTACCCGATAGAGTTCGGTGCACCGTCAAAACTTTGGTGTCGGCTACTGTAGTAGCTGAGGGACAAACAGGAGAGGGACGAACCGAGGTAGAAGTCGG orf 1 > N M G Y L K P R G S F E T T A D D I I D S L F V L S L L G S I F S L Age1 SpDon Rsr2 | Bpm1| | | || TGTCTGTGATTGCTGCGGACCGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTAT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 ACAGACACTAACGACGCCTGGCCATGAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATA orf 1 > S V I A A D R Y F T I F Y A L Q Y H N I M T V K R V G I I I S C I SpDon | CTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGA orf 1 > W A A C T V S G I L F I I Y S D S S A V I I C L I T M F F T M L A BspLU SpDon Eci1 | | | CTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGT orf 1 > L M A S L Y V H M F L M A R L H I K R I A V L P G T G A I R Q G A N Apa1 PspOM | BseY1 | | Ssp1 | | | | ATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 TATACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGACCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGT orf 1 > M K G A I T L T I L I G V F V V C W A P F F L H L I F Y I S C P Q SpDon BspE1 | | GAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 CTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCA orf 1 > N P Y C V C F M S H F N L Y L I L I M C N S I I D P L I Y A L R S SpAcc Xmn1 T7Ter Stu1 | | | | CAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAAATGGGGACAGAGCACGCAA 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 GTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTATAATTTACCCCTGTCTCGTGCGTT orf 1 > Q E L R K T F K E I I C C Y P L G G L C D L S S R Y EcoRV Not1 Xho1 EcoR1 Pst1 | BstX1 Eag1BsrB1| Xba1 | | | | | || | TATAGAAGCCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGA 1001 ---------+---------+---------+---------+---------+----1055 ATATCTTCGGCTTAAGACGTCTATAGGTCGTGTCACCGCCGGCGAGCTCAGATCT

PAGE 273

273 APPENDIX C HISTOGRAMS OF FACS CHIMERA DATA Histogram statistics were calculated rela tive to the isotype control as background. M1 = No label and isotype control peaks. M2 = Surface and total expression, peak only. M3 = Surface and total expression peaks + low end. M4 = Surface and total expression peaks + high and low ends. Color Key: Purple (filled) peak = No label Green = Isotype control Pink = Surface labeled with Anti-Flag-APC Total = Surface and intracellula r labeled with Anti-FLAG-APC

PAGE 274

274 Figure C-1. Flag-hMC2R in HEK cells FACS Histogram

PAGE 275

275 Figure C-2. Chimera 2C1 in HEK cells FACS Histogram

PAGE 276

276 Figure C-3. Chimera 2C2 in HEK cells FACS Histogram

PAGE 277

277 Figure C-4. Chimera 2C3 in HEK cells FACS Histogram

PAGE 278

278 Figure C-5. Chimera 2C4 in HEK cells FACS Histogram

PAGE 279

279 Figure C-6. Chimera 2C5 in HEK cells FACS Histogram

PAGE 280

280 Figure C-7. Chimera 2C6 in HEK cells FACS Histogram

PAGE 281

281 Figure C-8. Flag-hMC4R in HEK cells FACS Histogram

PAGE 282

282 Figure C-9. Chimera 4C1 in HEK cells FACS Histogram

PAGE 283

283 Figure C-10. Chimera 4C2 in HEK cells FACS Histogram

PAGE 284

284 Figure C-11. Chimera 4C3 in HEK cells FACS Histogram

PAGE 285

285 Figure C-12. Chimera 4C4 in HEK cells FACS Histogram

PAGE 286

286 Figure C-13. Chimera 4C5 in HEK cells FACS Histogram

PAGE 287

287 Figure C-14. Chimera 4C6 in HEK cells FACS Histogram

PAGE 288

288 Figure C-15. Flag-hMC2R in OS3 cells FACS Histogram

PAGE 289

289 Figure C-16. Chimera 2C1 in OS3 cells FACS Histogram

PAGE 290

290 Figure C-17. Chimera 2C2 in OS3 cells FACS Histogram

PAGE 291

291 Figure C-18. Chimera 2C3 in OS3 cells FACS Histogram

PAGE 292

292 Figure C-19. Chimera 2C4 in OS3 cells FACS Histogram

PAGE 293

293 Figure C-20. Chimera 2C5 in OS3 cells FACS Histogram

PAGE 294

294 Figure C-21. Chimera 2C6 in OS3 cells FACS Histogram

PAGE 295

295 Figure C-22. Flag-hMC4R in OS3 cells FACS Histogram

PAGE 296

296 Figure C-23. Chimera 4C1 in OS3 cells FACS Histogram

PAGE 297

297 Figure C-24. Chimera 4C2 in OS3 cells FACS Histogram

PAGE 298

298 Figure C-25. Chimera 4C3 in OS3 cells FACS Histogram

PAGE 299

299 Figure C-26. Chimera 4C4 in OS3 cells FACS Histogram

PAGE 300

300 Figure C-27. Chimera 4C5 in OS3 cells FACS Histogram

PAGE 301

301 Figure C-28. Chimera 4C6 in OS3 cells FACS Histogram

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334 BIOGRAPHICAL SKETCH Krista Renner Wilson was born in Ocala, Fl orida in 1981 to Robert and Janice Renner. Krista attended St. John Lutheran Sc hool in Ocala from kindergarten through 12th grade. At the age of eight Krista joined the 4-H Club, which quickly became a ve ry important part of her life and education. She raised and showed lambs and hogs and also developed a flourishing rabbitry. Through 4-H Krista learned showmanship, respons ibility, record-keeping and business skills which have been very valuable in academics a nd life. Krista graduated high school in 1999 with highest honors and received multiple scholarships for college. For 2 years, Krista attended Central Florida Community College in Ocala, FL where she majored in biology. Krista graduated in 2001 summa cum laude from the Honors Program and was also awarded the prestigious Science Student of the Year Award in 2001. Krista then traveled to Gainesville, FL to the University of Florida where she majored in animal science with a specialization in animal biology and a minor in chemistry. During the unde rgraduate years Krista was a part of the University Scholars Program where she worked with Dr. Carrie Haskell-Luevano to gain research experience. After graduation in 2003 w ith highest honors and receiving the J. Wayne Reitz Award given to the Outstandi ng Senior in the College of Agriculture and Life Sciences, Krista was married to Brandon Wilson and settled down in Gainesville to earn her doctorate in medicinal chemistry continuing to work with Dr. Carrie Haskell-Luevano. Krista was supported by the University of Florida Alumni Fellowship. She received her Ph. D. in 2008 and plans to take time off to raise her children before pursuing a career in academia.