Citation
Design, Synthesis, and Characterization of Peptides and Peptidomimetics for Mouse Melanocortin Receptors

Material Information

Title:
Design, Synthesis, and Characterization of Peptides and Peptidomimetics for Mouse Melanocortin Receptors
Creator:
JOSEPH, CHRISTINE G. ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Agonists ( jstor )
Amino acids ( jstor )
Biochemistry ( jstor )
Ligands ( jstor )
Melanocortin receptors ( jstor )
Melanocortins ( jstor )
Obesity ( jstor )
Receptors ( jstor )
Resins ( jstor )
Truncation ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Christine G. Joseph. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
12/31/2005
Resource Identifier:
71300991 ( OCLC )

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DESIGN, SYNTHESIS, AND CHARACTERIZATION OF PEPTIDES AND PEPTIDOMIMETICS FOR MOUSE MELANOCORTIN RECEPTORS By CHRISTINE G. JOSEPH 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 2004

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This dissertation is dedicated to my family for all of their suppor t and encouragement.

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iii ACKNOWLEDGMENTS I would like to express my sincere gratitude to all of my present and former colleagues in the Haskell-Luevano lab, for making my time there enjoyable. Special thanks go to Dr. Carrie Haskell-Luevano, my adviser, for all of her help and support throughout my graduate studies. I also thank Dr. Andrzej Wilczynski, who taught me peptide chemistry; and Dr. Xiang Simon Wa ng, who did the modeling in collaboration with us. Thanks go to Jim Rocca for all th e help obtaining and analyzing my proton nuclear magnetic resonance (1H-NMR) spectras. I thank the National Institutes of Health (NIH) for the minority supplementary gran t DK57080 that supported my work over the last 4 years. Most of all, I give very sp ecial thanks to my family, for all of their support and encouragement throughout my graduate studies.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABBREVIATIONS..........................................................................................................xii ABSTRACT....................................................................................................................... xv CHAPTER 1 INTRODUCTION........................................................................................................1 Obesity........................................................................................................................ ..1 Components of the Melanocortin System.....................................................................4 Melanocortin Receptors................................................................................................6 Melanocortin Endogenous Ligands..............................................................................9 Proopiomelanocortin Prohormone-Derived Agonists...........................................9 Endogenous Antagonists.....................................................................................11 The Melanocortin System and Energy Homeostasis..................................................13 2 GENERAL METHODOLOGIES..............................................................................17 Solid-Phase Peptide Synthesis....................................................................................17 The Merrifield or Boc Synthetic Strategy...........................................................19 Fmoc Synthetic Strategy......................................................................................21 Coupling Methods...............................................................................................23 Carbodiimides..............................................................................................24 Onium salts...................................................................................................27 Monitoring Procedures........................................................................................28 Purification and Analysis....................................................................................31 Development of Peptidomimetics...............................................................................32 General Strategies for the Deve lopment of Peptidomimetics.....................................33

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v 3 STRUCTURE-ACTIVITY RELATIONSHI P STUDIES OF MONOCYCLIC AGRP AT THE MOUSE MELANOCORTIN RECEPTORS...............................................36 Introduction.................................................................................................................36 Elongation of Yc[CRFFNAFC]Y...............................................................................38 Results........................................................................................................................ .40 Chemical Synthesis and Characterization...........................................................40 Biological Evaluation..........................................................................................40 Mouse melanocortin-1 receptor...................................................................41 Mouse melanocortin-3 receptor...................................................................41 Mouse melanocortin-4 receptor...................................................................43 Discussion...................................................................................................................45 Stereochemical Inversion of Arg111-Phe112-Phe113 Positions......................................47 Results........................................................................................................................ .48 Chemical Synthesis and Characterization...........................................................48 Pharmacology......................................................................................................49 Mouse melanocortin-1 and -5 receptors.......................................................49 Mouse melanocortin-3 and -4 receptors.......................................................51 Modeling into the mMC4R.................................................................................51 Discussion...................................................................................................................52 Modeling into the mMC4R.........................................................................................55 Summary.....................................................................................................................58 4 TRUNCATION STUDIES OF GAMMA2-MELANOCYTE-STIMULATING HORMONE................................................................................................................59 Introduction.................................................................................................................59 2-MSH SAR Studies..................................................................................................60 Alanine and D-amino Acid Scans.......................................................................61 Truncation of 2-MSH.........................................................................................62 Results........................................................................................................................ .62 Peptide Synthesis.................................................................................................62 Biological Evaluation..........................................................................................65 Mouse melanocortin-1 receptor...................................................................65 Mouse melanocortin-3 receptor...................................................................66 Mouse melanocortin-4 receptor...................................................................68 Mouse melanocortin-5 receptor...................................................................69 Discussion...................................................................................................................72 Modification of Met3 and Trp8 Side Chain........................................................72 C-terminal Truncation.........................................................................................74 N-terminal Truncation.........................................................................................75 Amidation and Acetylation of His-Phe-Arg-Trp.................................................75 Summary.....................................................................................................................77

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vi 5 STRUCTURE-ACTIVITY STUDIES OF UREA PEPTIDOMIMETICS AT THE MOUSE MELANOCORTIN RECEPTORS..............................................................79 Introduction.................................................................................................................79 SAR of Nonpeptide Ureas Based on a Linear Tripeptide...........................................84 Results........................................................................................................................ .86 Chemical Synthesis and Characterization...........................................................86 Biological Evaluation..........................................................................................87 Mouse melanocortin-1 receptor...................................................................90 Mouse melanocortin-3 receptor...................................................................91 Mouse melanocortin-4 receptor...................................................................91 Mouse melanocortin-5 receptor...................................................................92 Discussion...................................................................................................................93 Summary.....................................................................................................................95 6 STRUCTURE-ACTIVITY RELATI ONSHIPS OF THE MELANOCORTIN TETRAPEPTIDE Ac-His-DPhe-Arg-Trp-NH2 AT THE MOUSE MELANOCORTIN RECEPTORS: MODIFI CATION OF THE ARGININE SIDE CHAIN........................................................................................................................97 Introduction.................................................................................................................97 Truncation Studies...............................................................................................98 Alanine Scans......................................................................................................99 Melanocortin His-DPhe-Arg-Trp Tetrapeptide Sequence........................................100 Previous Modifications of the Arg8 Position in Melanocortin Receptor Ligands....101 Results and Discussion.............................................................................................103 Chemical Synthesis and Characterization.........................................................103 Biological Evaluation........................................................................................103 Summary...................................................................................................................110 7 EXPERIMENTAL....................................................................................................112 Peptide Synthesis......................................................................................................112 AGRP Elongation Peptides...............................................................................112 Method A: cyclization in solution...............................................................113 Method B: cyclization on resin...................................................................113 AGRP Stereochemistry Peptides.......................................................................114 Method A: disulfide bridge formation in solution.....................................117 Method B: disulfide bridge formation on resin .........................................117 -MSH Peptides................................................................................................118 Urea Analogues.................................................................................................120 Modified Tetrapeptide.......................................................................................121 Removal of aloc protecting group..............................................................122 Preparation of ureaor thi ourea-substituted tetrapeptides.........................123 Cell Culture and Transfection...................................................................................123 -Galactosidase Functional Bioassay.......................................................................124 NDP-MSH and AGRP(86-132) Iodination..............................................................125

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vii Receptor Binding Studies of AGRP Elongation Analogues.....................................126 Data Analysis............................................................................................................127 Computational Methods for AGRP Monocyclic Modeling......................................127 One-Dimensional 1H Nuclear Magnetic Re sonance Spectroscopy..........................128 Urea Analogues.................................................................................................128 Arg Modified Ureas, Thi oureas, and Carbamates..............................................128 8 CONCLUDING REMARKS....................................................................................130 Elongation of hAGRP(109-118) and Stereochemical St udies of hAGRP(103-122)131 Truncation of 2-MSH..............................................................................................133 Urea Nonpeptide Analogues.....................................................................................134 Structure-Activity Relationships of Melanocortin Tetrapeptides.............................134 APPENDIX A 1H-NMR FOR NONPEPTIDE UREA ANALOGUES............................................136 B 1H-NMR OF ARGININE-MODI FIED TETRAPEPTIDES....................................150 LIST OF REFERENCES.................................................................................................166 BIOGRAPHICAL SKETCH...........................................................................................186

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viii LIST OF TABLES Table page 3-1 Analytical data of the monocyclic AGRP peptides 1-8...........................................40 3-2 Pharmacology of the monocyclic AGRP peptides 1-8.............................................41 3-3 Binding results for AGRP derivatives 1-8...............................................................42 3-4 Analytical data of the monocyclic AGRP peptides with stereochemical conversion................................................................................................................49 3-5 Functional activity of monocycli c hAGRP(103-122) peptide analogues................50 4-1 Analytical data of 2-MSH analogues 16-43............................................................63 4-2 Functional activity of the 2-MSH analogues 16-43................................................64 4-3 Functional activity of the amidated a nd acetylated His-Phe-Arg-Trp tetrapeptide analogues..................................................................................................................76 5-1 Analytical data for tripeptide and urea analogues 47-66..........................................87 5-2 Functional activity of the tripep tide and urea nonpep tide analogues 47-66.............89 6-1 Analytical data for the Ar g modified tetrapeptides 67-97......................................104 6-2 Functional activity of the Ar g modified tetrapeptides 67-97.................................106

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ix LIST OF FIGURES Figure page 1-1 Obesity trends among US adults by state from 1985-2002........................................2 1-2 Melanocortin receptor system....................................................................................6 1-3 Comparison of wildtype (WT), MC3R knockout (MC3RKO) and MC4RKO mice........................................................................................................................... .8 1-4 Overview of POMC processing...............................................................................10 1-5 Sequence alignment of human Agouti and AGRP . ..................................................12 2-1 A general scheme for the preparati on of peptides by solid-phase peptide synthesis...................................................................................................................18 2-2 The Merrifield solidphase synthetic strategy..........................................................19 2-3 Possible mechanism for removal of the acid-labile butyloxycarbonyl (Boc)..........20 2-4 HF apparatus............................................................................................................21 2-5 The fmoc solid-phase synthetic strategy..................................................................22 2-6 Possible mechanism for base-catalylized removal of the N-terminal Fmoc ...........23 2-7 Possible mechanisms of activated amino acids racemization..................................24 2-8 Structures of widely used carbodiimides.................................................................24 2-9 Possible mechanisms of amide bond formation via carbodiimide activation..........25 2-10 A possible mechanism of active es ter formation using carbodiimides....................26 2-11 General structure of pho sphonium and aminium salts.............................................27 2-12 Structures of the most common aminium and phosphonium salts...........................28 2-13 Amino acid activiation with HBTU coupling reagent.............................................29 2-14 Possible mechanism of ruhemann’s purple formation.............................................30

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x 2-15 Possible structure of chloranil reaction product.......................................................31 2-16 The two colorimetric assays us ed to monitor peptide synthesis..............................31 2-17 Rational strategy for the deve lopment of peptidomimetics......................................33 3-1 Disulfide arrangement of the Cterminal domain of human AGRP.........................37 3-2 Model of the monocyclic hAGRP(103-122)-mMC4R complex..............................54 3-3 Graph of functional activity of th e peptides 5, 9, and 11 at the mMC4R................56 3-4 Schematic Diagram of the mouse melanocortin-4 receptor . ....................................57 4-1 Graph of 2-MSH analogue 21 and 2-MSH at the mouse MC1R...........................66 4-2 Graph of 2-MSH analogue 21 and 2-MSH at the mouse MC3R...........................68 4-3 Graph of 2-MSH analogue 21 and 2-MSH at the mouse MC4R...........................71 4-4 Graph of 2-MSH analogue 21 and 2-MSH at the mouse MC5R...........................71 4-5 Illustration of mMC5 R selective peptide 17............................................................74 4-6 Comparison of 2-MSH versus analogues with substitution at the 3 and 8 position.....................................................................................................................75 4-7 Illustration of 2-MSH truncation results at the mouse melanocortin receptors......78 5-1 Illustration of the putative DPhe-Arg-T rp amino acids interaction with mMC1R..81 5-2 Potent and selective nonpeptide compounds for the melanocortin receptors..........83 5-3 Illustration of the nonpeptide urea template.............................................................86 5-4 Structures of monomers us ed in the urea nonpeptide library...................................88 5-5 Structure of equipotent potent anal ogue at the MC1 and MC4 receptors................95 5-6 Illustration of the tripep tide 52, and the nonpeptide, 58..........................................96 6-1 Structures used to replace Ar g in the tetrapeptide template...................................105 6-2 Comparison of urea and thiourea analogues..........................................................108 6-3 Comparison of phe nyl-substituted urea..................................................................109 6-4 Illustration of the mMC1R se lective modified tetrapeptide...................................111

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xi 7-1 Picture of the manual reaction vessel.....................................................................114 7-2 Picture of the Advanced Chemtech 440 MOS synthesizer....................................117 7-3 Schematic representation for urea synthesis using solid phase methodology........121 7-4 Scheme for Arg side chain modi fication using solid phase methodology.............123 7-5 Picture of the semi-automated Advanced ChemTech synthesizer.........................124

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xii ABBREVIATIONS Acm acetomidomethyl ACTH Adrenocorticotropin hormone AGRP Agouti-related protein Aloc allyloxycarbonyl BMI Body Mass Index Boc tert -butoxycarbonyl cAMP Cyclic Adenosine monophosphate CNS Central nervous system 3D Three dimensional DCC N,N-dicyclohexylcarbodiimide DCM Methylene chloride DIC N,N-diisopropylcarbodiimide DIEA diisopropylethylamine DMAP N,N-(dimethylamino)-pyridine DMF dimethylformamide Fmoc 9-fluorenylmethoxycarbonyl gHOAc Glacial acetic acid GPCR G-protein coupled receptor HBTU 2-(1-H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

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xiii HF Hydrogen fluoride HMPB-MBHA 4-[4-(hydroxymethyl)-3-methoxyphenoxy]butanoyl-pmethylbenzhydrylamine 1H-NMR Proton Nuclear Magnetic Resonance HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole Kg kilogram m meter MBHA 4-methylbenzhydrylamine MC1-5R Melanocortin-(1-5) receptor MC3RKO Melanocortin-3 receptor knockout MC4RKO Melanocortin-4 receptor knockout MeOH methanol -, -, -MSH Alpha-, beta-, gamma-mela nocyte-stimulating hormone Pbf 2,2,4,6,7-pentamethyldihydrobe nzofuran-5-sulphonyl PC Prohormone convertase POMC proopiomelanocortin RP-HPLC Reverse phase-high performance liquid chromatography RT Room temperature SAR Structure-activity relationship tBu tert -butyl TFA Trifluoroacetic acid THF tetrahydrofuran

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xiv TM Transmembrane Trt trityl WT wildtype Z benzyloxycarbonyl

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xv 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 DESIGN, SYNTHESIS, AND CHARACTERIZATION OF PEPTIDES AND PEPTIDOMIMETIC FOR MOUSE MELANOCORTIN RECEPTORS By Christine G. Joseph December 2004 Chair: Carrie Haskell-Luevano Major Department: Medicinal Chemistry In the United States, the prevalence of obes ity has risen at an epidemic rate over the past decade. Based on Body Mass Index (BM I) of 30 or greater, more than 60% of American adults are considered obese. Alt hough still viewed more as a cosmetic than a health problem by the general public, excess wei ght is a major risk factor. A variety of health problems are directly associated w ith being overweight a nd obese. Successful treatment of obesity and obe sity-related diseases require s an understa nding of the complex pathways involved in weight homeostasis and feeding behavior. New insights into the mechanisms that control body weight are providing an increasingly detailed framework for a better understanding of obesity pathogenesis. Over the last decade, genetic and pharmacological ev idence has emerged that supports a role of the melanocortin (MC) receptor system in weight homeostasis. Current data suggest that central MC3 and MC4 recepto rs, along with their endoge nous agonists and antagonist,

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xvi are key components responsible for regulati ng body weight via the modulation of both food intake and energy expenditure. This dissertation focuses on the design, synthesis and ch aracterization of ligands for the mouse melanocortin receptors. Four lib raries will be discussed that include (1) analogues of the MC3 and MC4 receptors anta gonist Agouti-related protein (AGRP); (2) truncation of 2-MSH, an agonist at the MC1 and MC35 receptors with slight selectivity for the MC3R; (3) modification at the Arg position of Ac-His-DPhe-Arg-Trp-NH2 the minimal sequence required for potent melanocor tin receptor agonist activity; and (4) the development of nonpeptide ligands ba sed on the tripeptide Phe-Trp-Lys-NH2. The results of these studies include the identification of several key structureactivity trends, receptor subtype selectivity pr operties, identification of residues in ligandreceptor interactions, and novel ligands with activities. Some of the results include the development of a novel agonist template when stereochemical inve rsion studies turned the hAGRP(103-122) from an antagonist into an agonist. Truncation of -MSH identified a shorter analogue with equi potent agonist activity at th e mMC1R and mMC3-5R. We identified a nonpeptide ligand with nanamola r agonist activity at the mMC4R that was selective over the mMC4 and mMC5 r eceptors. Finally, a novel mMC1R thiourea selective agonist was identified when Ac-His-DPhe-Arg-Trp-NH2 was modified at the Arg position. The details of these studie s are covered in this dissertation.

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1 CHAPTER 1 INTRODUCTION Obesity In the United States, obesity has risen at an epidemic rate during the past 20 years. Figure 1-1 shows the increase in obesity from when it was first recorded to the later report today.1 In 1985, only a few states were part icipating in the Center for Disease Control (CDC) Behavioral Risk Factor Su rveillance System (BRFSS) providing obesity data and no state was greater than 14%. In 1991, four states were reporting obesity prevalence rates of 15-19% and no states repo rted rates at or a bove 20%. In 1997, sixteen states had obesity rates of 1014%; thirty-one states had rate s of 15-19%; three states had rates >20% and no states had ra tes > 25%. In 2001, four years la ter, one state had rates of 10-14%; twenty-one states had rates of 15-19%; twenty-seven states had rates of 20-24%, and one state had rates of >25%. In 2002, one ye ar later, no states reported rates at or below 14%, 18 states had rates of 15-19%; 29 states had rates of 20-24%; and 3 states had rates over 25%.1-4 Obesity is characterized by an increase in adipose tissue mass.5,6 Evaluating the exact quantity of adipose tissue is rather difficult. However, in clinical practice, body fat mass is estimated by body mass index (BMI), or by waist circumference. Individuals are considered obese when thei r BMI is greater than 30.1,6 Research indicates that the situation is worsening rather than improvi ng. The obesity epidemic covered on television (TV) and in the newspapers di d not occur overnight. Overall, a variety of factors play a role in obesity, making it a complex health issue.

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2 19851991 MA RI CT NJ DE MD & DC ME VA NH VT NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA TX OK KS NM AZ NV CA UT CO NE SD ND MT WY ID OR WA AK HI KY MT ID OR WA NV MT ID OR WA AZ UT WY NM CO AZ UT WY CA NV MT ID OR WA TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY MA RI CT NJ MD & DC ME NH VT DE AK HI TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY CA AK HI NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DEHI TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TNW VOH IN MI WI IL AL MS AR MO IA MN LA KY CA AK NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DE No Data< 10%10%-14%15%-19%20%-24% 25% TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY CA AK HI NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DE19972001 2002 19851991 MA RI CT NJ DE MD & DC ME VA NH VT NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA TX OK KS NM AZ NV CA UT CO NE SD ND MT WY ID OR WA AK HI KY MT ID OR WA NV MT ID OR WA AZ UT WY NM CO AZ UT WY CA NV MT ID OR WA TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY MA RI CT NJ MD & DC ME NH VT DE AK HI TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY CA AK HI NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DEHI TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TNW VOH IN MI WI IL AL MS AR MO IA MN LA KY CA AK NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DE No Data< 10%10%-14%15%-19%20%-24% 25% TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY CA AK HI NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DE19972001 2002 MA RI CT NJ DE MD & DC ME VA NH VT NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA TX OK KS NM AZ NV CA UT CO NE SD ND MT WY ID OR WA AK HI KY MT ID OR WA NV MT ID OR WA AZ UT WY NM CO AZ UT WY CA NV MT ID OR WA TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY MA RI CT NJ MD & DC ME NH VT DE AK HI TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY CA AK HI NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DEHI TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TNW VOH IN MI WI IL AL MS AR MO IA MN LA KY CA AK NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DE No Data< 10%10%-14%15%-19%20%-24% 25% No Data< 10%10%-14%15%-19%20%-24% 25% TX OK KS NE SD ND NM CO AZ UT WY VA NY PA NC SC GA FL TN WV OH IN MI WI IL AL MS AR MO IA MN LA KY CA AK HI NV MT ID OR WA MA RI CT NJ MD & DC ME NH VT DE19972001 2002 Figure1-1. Obesity trends among US adu lts by state from 1985-2002 based upon figures provided by the center for dise ase control (CDC) website Obesity can increase the risk of health complications ranging from nonfatal to fatal debilitating conditions such as heart dis ease, diabetes, high blood pressure, stroke, osteoarthritis, sleep apnea, gallbladder disease, and some forms of cancer (uterine, breast, colorectal, kidney); to psychological conseque nces ranging from lowered self-esteem, to social isolation, to clinical depression. Se vere obesity is associated with a 12-fold increase in mortality in 25 to 35 year olds when compared to lean individuals. In the United States (US), at least one child in five is overweight and the number of overweight children continues to grow. Over the last two decades, this number has increased by more than 50%, and the numb er of extremely overweight children has

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3 nearly doubled.7 Although children have fewer wei ght-related health problems than adults do, overweight children are at high risk of becomi ng overweight adolescents and adults. Epidemiological studies have shown that th e prevalence for obesity increases at an alarming rate in the last 10 years. For exam ple, the prevalence of obesity jumped by about 10-50% in the US and most European countries. The prevalence of obesity in adults is 10-25% in most countries of Wester n Europe, and 20-25% in some countries in the Americas. Today, an estimated 300 million people around the world are obese; and prospective studies predict that th is number will continue to climb.8 In addition to the epidemic of obesity in the adult population, the prevalence of obesity in children is dramatically increasing as well. In 1999, 13% of children aged 6 to 11 years, and 14% of adolescents aged 12 to 19 years in the Unite d States were overweight. This prevalence has nearly tripled for adolescents in the past two decades. Overweight adolescents have a 70% chance of becoming overweight or obese ad ults. This increases to 80% if one or both parents are overweight or obese. The increase in obesity prevalence over the past two decades results in an increase in the cost of treating obesity and obes ity-related diseases. A recent study focused on state-level obesity-related medical expenditures estimated the total expenditure to be $75 billion based on Medicare and Medicaid covera ge, averaging approximately 6% of adult medical expenditures.9 This striking rise in obesi ty is attributed to a combination of genetic, social, behavioral factors, and to a profound change in our modern life style. Individuals may become obese if exposed to an environm ent where food is easily available and a

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4 sedentary life style is promoted. Genetic predisposition might accentuate this tendency.6 Mechanisms responsible for the developmen t of obesity were not well understood until recently. Numerous studies clearly establish the relationship between adipose tissue mass and the brain circuitry involve in regul ating energy homeostasis. Interestingly, hypothalamic centers in the brain have been f ound to play a critical role in regulating food intake and energy expenditure. These br ain centers can sense peripheral hormones involved in maintaining body weight (such as insulin and lepti n, and adipose tissuederived peptides). Furthermore, a number of hypothalamic expressed neuropeptides are believed to play a major role in regulating food intake.10 These neuropeptides are endogenous ligands for a number of G-protei n coupled receptors (GPCRs). Modulating the activity of GPCRs via pep tide binding has been shown to alter hypothalamic response to food intake. Components of the Melanocortin System The melanocortin system is one of ma ny pathways particip ating in feeding behavior, and weight and energy homeostasi s. The melanocortin-receptor family belongs to the G protein-coupled receptor (GPCR) supe rfamily (a class of receptors that mediate most cell-to-cell communi cation within humans).11 The broad variety of ligands (including hormones, neurotransmitters, ions and amino acids) that signal via GPCRs underscores the physiological importance of th is receptor class. Th e ability of small molecules to modulate the act ivity of GPCRs with a high degree of selectivity and sensitivity, combined with the anatomically se lective nature of GPCR gene expression, is why this receptor class is pharmaceutically important. A large percentage of today’s prescription drugs target one or more GPCRs, with most major therapeutic areas served

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5 to some extent by a number of GPCR-based drugs.12 Clearly, no single class of genes ranks higher than GPCRs in term s of drug-discovery potential. G protein-coupled receptors consist of seven hydrophobic transmembrane (TM) helices connected by three extr acellular loops and three intrac ellular loops. GPCRs differ in the length and function of their extracellular N-terminal, cytosolic C-terminal, and intracellular loops.13-15 Change in conformation of the TM-VI region is responsible for receptor activation.15,16 The switch from inactive to the active conformation is due to a rotation of TM-VI and a separation from TM-III, which unmasks G-protein binding sites.17-19 Receptor interaction with the G-protei n is followed by either activation or inhibition of a second messenger signal. Ligands that stimulate a receptor to initiate a functional response are called agonists. T hose that inhibit functional activity are antagonists. The melanocortin receptor family co nsists of five subtypes (MC1R-MC5R) identified and characterized to date.20-26 The five receptor subtypes at the amino acid level are about 42-67% identical and 75-94% similar among mammals for each receptor, with the MC4R being the most conserved and MC1R the least.27 The five G proteincoupled melanocortin receptors (MCRs) ar e all linked to cAMP generation via the stimulatory G protein Gs and adenylate cyclase (Figure 1-2). The endogenous agonists for the melanocortin receptors are derived by post-translational processing of the proopiomelanocortin (POMC) gene transcri pt. The two known endogenous antagonists of GPCRs, agouti and agouti-related protein ( AGRP), regulate the melanocortin receptors.

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6 COOH NH2Intracellular Extracellular AC GDP Gs GTP cAMP accumlationAgonist (ex. -, -, MSH, ACTH) I VI VII Antagonist Agouti, AGRP Melanocortin ReceptorsMC1R-Pigmentation MC2R-Steroidogenesis MC3R-Energy Homeostasis MC4R-Energy Homeostasis MC5R-Exocrine gland function L COOH NH2Intracellular Extracellular AC GDP Gs GTP cAMP accumlationAgonist (ex. -, -, MSH, ACTH) I VI VII Antagonist Agouti, AGRP Melanocortin ReceptorsMC1R-Pigmentation MC2R-Steroidogenesis MC3R-Energy Homeostasis MC4R-Energy Homeostasis MC5R-Exocrine gland function L Figure 1-2. Melanocortin Receptor System. Of pa rticular interest is the interaction of agonist ligand (red circle in center of transmembrane domain) leading to cAMP accumulation through adenylate cycl ase activation and the antagonist (brown horseshoe) which does not activate adenylate pathway. Melanocortin Receptors The MC1R was the first of the melanocorti n receptor gene family to be cloned by Mountyjoy et al.25 from mouse melanoma cell line a nd primary culture of normal human melanocytes, and by Chhajlani and Wikberg21 from human melanoma cells. Human MC1R has a high and almost equal affinity for -MSH and ACTH, less affinity for -MSH, and the lowest affinity for -MSH.21,28 Human and mouse MC1R share 76% identity in amino-acid sequence,29 but mouse MC1R has a much lower affinity for ACTH than -MSH, and -MSH is more potent at the hMC1R than at the mMC1R. The MC1R is expressed by cutaneous melanocytes, wh ere it has a key role in determining skin and hair pigmentation. However, other cell types in the skin also express MC1R, including

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7 keratinocytes, fibroblasts, endothelial cells, and antigen-presenting cells.30 Other tissues and cell types have also been found to express MC1R.31 In this respect, it is notable that MC1R is expressed by leukocytes, where it mediates the anti-inflammatory and immunomodulatory properties of melanocortins. MC2R was cloned shortly after th e MC1R from the adrenal gland.20,25 MC2R is the ACTH receptor, and is expressed in the adre nal cortex at the site for glucocorticoid production,25 where it mediates the effects of AC TH on steroid secretion. MC2R is expressed in the adipocytes, and it mediates most of the lipoly tic activity of ACTH.28 MC2R is distinguished pharmacologically from the other MCR subtypes in that it is activated only by ACTH, and has no affinity for -, -, or -MSH. A rare human autosomal recessive disorder, hereditary isolated glucocor ticoid deficiency, is caused by mutations in MC2R.32 Notably, MC2R is also expressed by adipose tissue in mice and humans.33 Although ACTH is lypolytic in mice, it is not so in humans; and the function of MC2R in human adipose tissue is presently unclear. MC3R was the third MCR to be cloned and the human MC3R is 361 amino acids long.22,26,34 MC3R shows similar binding affinity for -, -, -MSH, and ACTH. The MC3R is expressed in many areas of the cen tral nervous system (CNS) such as the hypothalamus, hippocampus, anterior amygdala , and cortex, suggesting a role in thermoregulation, and regulation of cardiovascular functions.28 The MC3R is also expressed in several peripheral tissues, including the gastrointestinal tract, heart, and placenta; and is detectable in the test is, mammary gland, skeletal muscle, and kidney.22,28,31,35 The MC3R is implicated as having a role in energy homeostasis, because

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8 MC3R knockout mice have been characterized by increased fat mass accompanied by decreased lean mass (Figure 1-3).36,37 Human MC4R consisting of 333 amino aci ds was the second neural MCR to be cloned.23 It is expressed widely in the central nervous sy stem (CNS), including the cortex, hypothalamus, thalamus, and brain stem.23 The distribution of the receptor suggests its involvement in autonomic a nd neuroendocrine functions. The order of potency of the endogenous melanoc ortin ligands at the MC4R is, -MSH = ACTH > MSH >> -MSH.27,28 The MC4R is involved in en ergy homeostasis and feeding behavior.38,39 This was substantiated by the fact that MC4R knockout mice were severely obese, hyperphagic, and developed hyperins ulinemia and hyperleptinemia (Figure 1-3).38,40 The role of MC4R in feeding behavior was determined after administration of the synthetic agonist MT-II did not redu ce food intake in MC4R knockout mice.38,39 The MC4R has also been found to play a role in erectile function.41 Figure 1-3. Comparison of wildtype (WT), MC3R knockout (MC3RKO) and MC4RKO mice. The MC4RKO mouse is hyperphagi c and weighs significantly more than both the MC3RKO and the WT mice. The MC3RKO is not as obese as the MC4RKO and is believed to have increased fat mass and reduced lean mass.

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9 The MC5R was the last of the melanocort in receptor gene family to be cloned.24,42 MC5R is expressed at high levels in the exocrine glands, incl uding the lacrimal and harderian glands.43 It is also expressed in skeletal muscle and skin tissues; particularly in the sebaceous glands, where it may play a role in sebaceous-gland lipid production.31,43-45 Low levels of expression are also detected in the brain.31,42-45 The order of potency of the melanocortins in activating the MC5R is -MSH > ACTH = -MSH >> -MSH.27,28 Functions of MC5R are still poorly understood, and are specul ated to include gastric and anti-inflammatory effects, and re gulation of aldosterone secretion.24,42,44,46 The role of MC5R in exocrine secretion has th e potential to be exploited for treating skin disorders such as acne.47 Melanocortin Endogenous Ligands Proopiomelanocortin Prohormone-Derived Agonists Human Proopiomelanocortin (P OMC) is a 267-amino-acid pr ecursor protein that is synthesized in the pituitary; in the arcu ate nucleus of the hypothalamus; in the nucleus of the solitary tract in the brain stem; and in several peripheral tissues including the urinary tract, gastrointestinal tract, adrenal, spleen, lung, and thyroid, and in cells of the immune system.33 Posttranslational processing of POMC is tissue-specific, and results in the production of a number of peptides with very different biological activities (Figure 1-4).48-50 Functionally active peptides ar e produced by endoproteolytic cleavage at adjacent pairs of basic amino acids, with prohormone convertases PC1 and PC2.51,52 In the anterior pituitary, POMC is processed predominantly to adrenocorticotropin (ACTH), which is critical for maintaining adrenocortical function; to -lipotropin (LPH), and a 16-kDa N-terminal fragment. In the hypothalamus and in the intermediate lobe of the pituitary,

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10 POMC is more extensively proc ess, where ACTH is further process to produce melanocyte-stimulating hormone ( -MSH) and corticotropin-like intermediate lobe peptide (CLIP); -LPH is process to -endorphin and -LPH which is further process to -MSH; and N-terminal POMC is process to 3-MSH which is further process to 1-MSH and 2-MSH (Figure 1-4). -endorphin 1-31 -endorphin 1-27 ACTH SYSME HFRW GKPVGKKRRPVKYPNGAEDESAEAFPLEF-OH -MSH Ac-SYSME HFRW GKPV-NH22-MSH YVMG HFRW DRFG-OH-MSH AEKKDEGPYRME HFRWGSPPKD-OH pre-POMC Signal sequence pro-MSH Joining Peptide ACTH -LPH N-POMC 1-49 3-MSH -MSHCLIP -LPH -261241RK RR KR KR KKRR KR KK KR KR N-LPH -MSH 1-MSH PC1 PC2P C 2 , C P E , P A M , N A TPC1 PC1PC1 PC2 PC2 PC2 PC2 PC2 2-MSH -endorphin 1-31 -endorphin 1-27 ACTH SYSME HFRW GKPVGKKRRPVKYPNGAEDESAEAFPLEF-OH -MSH Ac-SYSME HFRW GKPV-NH22-MSH YVMG HFRW DRFG-OH-MSH AEKKDEGPYRME HFRWGSPPKD-OH pre-POMC Signal sequence pro-MSH Joining Peptide ACTH -LPH N-POMC 1-49 3-MSH -MSHCLIP -LPH -261241RK RR KR KR KKRR KR KK KR KR N-LPH -MSH 1-MSH PC1 PC2P C 2 , C P E , P A M , N A TPC1 PC1PC1 PC2 PC2 PC2 PC2 PC2 2-MSH Figure 1-4.Overview of POMC processing by prohormone convertases into smaller proteins and the primary sequences of the melanocortin peptides. The minimal sequence required for binding and activa tion of melanocortin receptors, also called the message sequence in the prim ary structures of the melanocortin peptides are depicted in bold. Abbrev iations: POMC, Pro-opiomelanocortin; ACTH, adrenocortiotropic hormone; MSH, melanocyte stimulating hormone; LPH, lipotropic hormone; CLIP, corticotropin-like intermediate lobe peptide; PC, prohormone convertases; CPE, carboxypeptidase E; PAM , peptidyl amidating mono-oxygenase (amidation); N-AT , N-acetyltranferase (acetylation).

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11 The POMC prohormone also pr oduces the opiate peptide -endorphin (hence the name pro-opio-melanocortin). Among the peptide products of POMC, the melanocortins all share the central amino acid sequence His-Phe-Arg-Trp, which is a key pharmacophore needed for the biological activity of these pept ides (Figure 1-4).53-56 Rare mutations in the POMC hormone and PC1 have been found in humans, and are associated with adrenal insufficiency and early-onset obesity and, in the case of POMC mutations, altered pigmentation.57,58 Endogenous Antagonists Perhaps one of the most intere sting aspects of the melanocor tin system is that it has two endogenous antagonists: agouti and agouti-related protein (AGRP) (Figure1-5). These proteins are unique in that no other endogenous inhibitory proteins have been identified for any of the seven-transmembrane receptor families. Agouti and AGRP are paracrine signaling molecules, which are endogenous antagonist s of the MC1R, MC3R and MC4R.59 Of physiological significance, agouti and AGRP have MCR subtype selectivity.60,61 Interestingly, agouti and AGRP both have a cysteine-rich COOH-terminal domain. Nuclear magnetic resonance (NMR) studies show that the cysteine residues in AGRP adopt a structural motif called an inhibitor cystine knot.62 This motif is common to invertebrate toxins; but in mammals, this struct ure is unique to AGRP (Figure 1-5). AGRP has been shown in vitro to be an inverse agonist.63,64 Thus AGRP has the potential, in vivo, to regulate MCRs, even in the absence of melanocortins. Agouti is the name of an animal/rodent th at has a hair-color pattern characterized by a subapical yellow band on an otherwise black or br own background. Historically, scientific interest in the agou ti locus extended beyond its effect on coat color. Dominant

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12 mutations of the agouti gene cause mice to develop yellow fur, obesity, insulin resistance, increased somatic growth, and a predispostion to tumorigenesis.65 With the isolation of the gene encoding agouti, it was noted that these pleiotropic effects were associated with deregulated expression of agouti in all tissues.65 Later investigations showed that the obesity displayed by these muta nt mice is secondary to the ectopic expression of agouti in the hypothalamus, where it acts as an antagonist of -MSH at MC4R.66 In light of recent discoveries that hypothalamic -MSH is a major satiety factor that transmits its message by activating MC4R, the hyperphagia and resultant obesity of those animals are readily understood. AGRP MLTAALLSCALLLALPATRGAQMGLAPMEGIRRPDQALLPELPGLGLRAPLKKTTAEQAEEDLLQEAQALAE Agouti MDVTRLLLATLLVFLCFFTANSHLPPEEKLRDDRSLRSNSSVNLLDVPSVSIV ALNKKSKQIGR K -AAEKKRSSKKE * * ** * * * * * * AGRP VLDLQDREPRS S RR CV RLHE SC LGQQVP CCDPCA T C Y C RFF NAF C Y CR K L GTAMNP C SRT Agouti ASMKKVVRPRTP L S AP CV ATRN SC KPPAPA CCDPCA S C Q C RFF RSA C S CR V L SLN C Figure 1-5. Sequence alignment of human Agouti and AGRP . Identical amino acid residues are shown in red. Of particul ar interest for agouti/AGRP structure and function are the 10 Cysteine residues (mark by asteriks) and the conserved RFF triplet in the C-terminal domain (blue letters). The normal role of agouti, however , is to act in conjunction with -MSH and MC1R to determine mammalian coat color. Agouti is produced by the dermal papillae cell and acts on the adjacent melanocyte to block melanocor tin action at MC1R. This interaction has a major effect on pigmentation. Pharmacologically, agouti is a high-affinity, competitive antagonist of the melanocortin peptides at the MC1R and MC4R. In rodents, agouti is expressed only in skin. The human homolog of agouti, called agouti-signaling protein (ASIP), has a wider pattern of expression, including adipose tissue,

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13 testis, ovary, and heart; and lower levels of expression in foreskin, kidney, and liver.60 However, humans do not have a banded agouti-like hair pattern, and the role of ASIP in hair and skin pigmentation in humans is doubtful. Currently, the physiological function of ASIP in humans is unknown. After the discovery of agouti, AGRP was identified by database searches for molecules with homology to agouti.67 AGRP is an equipotent competitive antagonist of MC3R and MC4R.68 AGRP is expressed primarily in the arcuate nucleus of the hypothalamus, the subthalamic re gion, and the adrenal cortex; with a small amount of expression observed in the lung and kidney.59,69,70 However, its major physiological function is in the hypothalamus, where AGRP acts as a potent orexigenic (appetitestimulating) factor due to its ab ility to antagonize me lanocortins at MC3R and MC4R.59 The Melanocortin System and Energy Homeostasis Studies firmly implicated melanocortins in the inhibition of food intake on the basis of the observation that injecting ACTH (1-24) into th e lateral ventricle or ventromedial hypothalamic nucleus of the brain inhibited food intake in rats,71,72 and that POMC mRNA levels were regulated by metabolic state.73 However, in 1994, researchers took greater notice of the melanocortin system as a me diator of feeding behavior. By that time, the MCRs had been cloned, and it was known that MC3R and MC4R were expressed in the hypothalamus (a CNS region that controls many physiological functions, including feeding behavior). Importantly, that year , it was discovered that agouti was a potent antagonist of MC4R.66 It was hypothesized that obesity of mice with dominant mutations of the agouti gene was due to over-expression of agouti in the hypothalamus and its antagonism of MC4R. Several Studies, in 199740,67,74 solidified these observations into a coherent framework. First, it was shown that the MC4R antagonist SHU-9119 could

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14 block the inhibition of food intake i nduced by the nonspecific melanocortin agonist MTII.74 Second, targeted deletion of MC4R resulted in obesity and hyperphagia.40 Finally, the endogenous agouti-like orexigenic factor AGRP was discovered.67 These observations set the stage for a multitude of studies that establish the hypothalamic melanocortin system (MC4R, POMC peptides, and AGRP) as one of the convergence point for peripheral and central factors regulating feeding behavior and metabolism. Although -MSH is presumed to be the most relevant melanocortin involved in energy regulation in the hypothalamus, PO MC neurons release a complex mixture of POMC peptides believe to be involve in energy regulation.75 More recently, the MC3R was shown to be involved in energy homeostasis. MC3R-null mice resulted in reduced lean body mass, and increased subcutaneous fat, while maintaining a relatively normal body weight.36,37 Notably, the aforementioned observations ex tend to humans. It has been estimated that MC4R mutations occur in 4% of severely obese French individuals76,77 most occurring through a frameshift mutation re sulting in a nonfunctional receptor. Later studies identified many additional hetero zygous mutations in human MC4R gene.76,78-85 The hypothalamic melanocortin system is involved in obesity, and has also been implicated in cachexia86 and anorexia87 in rodents. The melanocortin system’s role in feeding behavior makes it an attractive target for the development of antiobesity agents. This is particularly true for the MC3R and MC4R. Many academic and pharmaceutical laboratories focus on identifying potent and selective melanocortin ligands to understand structural and functional char acteristics, with most of the focus on MC4R. Our study involved four ma in aspects of the design, synthesis, and

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15 characterization of melanocortin receptor ligands. The first asp ect (Chapter 3), details the structure-activity relationshi p (SAR) studies of a series of 15 analogues based on the Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr decapeptide sequence of the endogenous antagonist, AGRP that was shown to be the mi nimal sequence required for binding to the MC3R and MC4R.68,88 Although this sequence binds to the MC3R, it ha d no functional activity at this receptor. We hypothesized that elongation of this sequence should reestablished MC3R antagonism. The aim of the SAR was to identify the amino acids necessary for MC3R antagonism. This was fo llowed by stereochemical inversion of the Ag-Phe-Phe triplet common to the ASIP and AGRP endogenous antagonists needed for them to recognize and antagonize the melanocortin receptors. The second aspect (Chapter 4) was truncation of 2-MSH (Tyr-Val-Met-Gly-HisPhe-Arg-Trp-Asp-Arg-Phe-Gly-OH). The 2-MSH peptide preferentially activates the hMC3R. Because of the MC3R involvement in energy homeostasis, 2-MSH may be an excellent starting point to help design MC3R selective ligands. Ou r aim was to perform truncation of 2-MSH to identify the minimal active fragment and the important amino acid residues necessary for agonist activ ity at the melanocortin receptors. The third aspect (Chapter 5) was identifying nonpeptide ligands for the melanocortin receptors. Using a rational a pproach, we designed, synthesized, and characterized a series of 14 urea analogue s based on the tripeptide Phe-Trp-Lys-NH2. Our aim was to identify nonpeptide ligands with improved potency and selectivity compared to the peptide, since nonpeptides genera lly have more favorable pharmacokinetic properties compared to peptides.

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16 The fourth aspect (Chapter 6) was SAR study of a series of thirty-one analogues based on the Ac-His-DPhe-Arg-Trp-NH2 tetrapeptide with modifications at the Arg position. Many studies show that the Arg residue is required for ligand potency while a number of selective nonpep tide ligands for the MC1R a nd MC4R didn’t require the presence of this basic moiety for receptor selectivity. Our aim was to design a novel series of analogues with side-chain modifica tion at the Arg position, to identify trends that result in increase or decrease in poten cy and receptor selectivity at the melanocortin receptors.

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17 CHAPTER 2 GENERAL METHODOLOGIES Solid-Phase Peptide Synthesis Bruce Merrifield89 revolutionized the pe ptide chemistry field in 1963. He described the complete synthesis of a tetrapeptide teth ered to an insoluble polymeric support. The Merrifield method involved att aching the C-terminal amino aci d of a target peptide to a solid support, and then elongating the pep tide chain with suc cessive condensation reactions. The original synthe sis led to several byproducts, but the synthetic scheme was improved on; eventually leading to the synthesi s of a nine-residue pep tide with yields and purity much better than what could be accomplished with solution chemistry.90,91 Merrifield’s method was a significant improve ment over the conventi onal strategies of peptide synthesis that used solutionphase methodology. Solution-phase peptide syntheses were time consuming because afte r every coupling reacti on the intermediate had to be isolated, purified (some cases by cr ystallization) and characterized before the next coupling reaction.89 With the solid-phase approach, isolation and purification after each step was accomplished by washing and filt ering, hence, eliminating the exhaustive isolation, purification, and characterization steps required in solution chemistry. Eliminating the extra steps enhanced bot h the speed of synthesis, purity, and characterization is done only at the end of the synthesis. The solid-phase synthetic strategy is illust rated in Figure 2-1. The N-terminus of amino acids is protected with a “temporary” group that is re moved prior to addition of the

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18 next amino acid. Reactive side chain func tionalities are blocked with “permanent” protecting groups that are not remove d until the synthesis is complete.89 Deprotection of temporary protecting group -amino acid protected amino acid Activation CouplingPeptide Synthesis CycleSolid support Protected peptide Cleaveage & Deprotection Crude Peptide Figure 2-1. A general scheme for the prepar ation of peptides by solid-phase peptide synthesis. The C-terminal of the amino acid is activ ated with a suitable coupling reagent before coupling to the free amino group of the re sin. The first step is attachment of the C-terminal amino acid in the peptide sequence to the polymeric support or resin . Amino acids are generally introduced in excess to insure the reaction is driven to completion so that there will be less chances of obtaini ng truncated or elonga ted analogues. Once attached to the resin, the reagents an d excess amino acid are simply removed by successive filtration and washing steps. Following attachment and washing, the temporary N-terminal protecting group is re moved and introduction of the next amino acid is ready to proceed. The next amino acid is introduced and coupled to the preceding amino acid via amide bond formation. The te mporary N-terminal protecting group is removed and the cycle is ready to be repeat ed with the next amino acid. Once the desired

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19 sequence is reached, the peptide is liberated from the solid support by cleavage of the linker and the permanent or thogonal protecting groups are removed in one step. The Merrifield or Boc Synthetic Strategy The method of solid-phase pe ptide synthesis originally developed by Merrifield is still used today in many laboratories across the globe with little modification (more polymeric support and side-chain protecting gr oups available). The synthetic strategy makes use of amino acids protected primarily with t -butyl and benzyl derivatives. Figure 2-2 shows the Merrifield synthetic strategy. N-terminal t -butyl groups are used for temporary protection and are selectively re moved prior to each amino acid coupling cycle. Benzyl-based groups are used for more permanent protection of reactive amino acid side chains and are removed when synt hesis of the desired peptide oligomer is complete. OH O H N O H2N O HN O O ClHF HF TFAAcid-labile Temporary Protection Group Acid-labile Permanent Protection Groups Acid-labile peptide-resin linker Insoluble Polymeric SupportO O O O Figure 2-2. The Merrifield So lid-Phase Synthetic Strategy The most common t -butyl protecting moiety us ed in this strategy is the t -butyloxycarbonyl (Boc) group, referred to as “Boc chemistry methodology” which was the approach developed by Merrifield. The Bo c group is selectively removed under acidic conditions from the N-terminus with trifluor oacetic acid (TFA) solutions. Once the Boc

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20 group is removed the protonated amino terminus remains as a trifluoroacetate salt which is neutralized with a tertiary amine such as diisopropylethylamine (DIEA), before continuation of condensation with the next amino acid. A possible mechanism of Boc removal is shown in Figure 2-3. F F F O O ON H O O H ON H O O H N O H H ON H O O F F F O O HCO2+ + + Figure 2-3. Possible mechanism for removal of the acid-labile butyloxycarbonyl (Boc) amino protection group from resin-bound alanine. After activation of the carboxy terminus of the next protected amino acid, extension of the peptide chain is achiev ed by coupling it to the free amino terminus on the resin or polymeric support. Liquid hydrogen fluoride (HF) , a very strong acid, is used to remove the orthogonal protecting groups from reactive amino acid side chains and to liberate the peptide from the solid support in a specialize apparatus (Figure 2-4) . These two steps are typically done simultaneously. Deprotection of the reactive si de chains usually generates reactive products of cationi c nature, which can react w ith the peptide generating unwanted side products.92 Amino acid side chains with electron-rich functional groups

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21 are particularly prone to modi fication by these cationic species.92 To avoid the unwanted side products, appropriate “scavenger(s)” are in corporated into the cleavage solution to quench the reactive intermediates generate d in the orthogonal deprotection/cleavage step.92 Scavengers are nucleophilic reagents su ch as water, phenol , thiols, and alkyl silanes. Due to the toxic and volatile na ture of HF, extreme caution and special equipment must be used during the orthogona l deprotection and cleavage steps of the Merrifield strategy or Boc methodology (Figure 2-4). HF HF Figure 2-4. The HF apparatus use for deprotecti on and cleavage of peptides from polymeric support or resin after “B oc” synthesis methodology Fmoc Synthetic Strategy The Boc strategy has significantly increased the speed and purity of peptide synthesis but unfortunately, it cont ained some shortcomings. Both N temporary and orthogonal permanent protecting groups are acid labile, resulting in an inherent loss of amino acid side chain protecting groups duri ng Boc removal. This loss generates two sources of reactive functionali ties, the amino acid side chai n and the reactive orthogonal protection group, thereby providing a means for si de reactions to occur. Some loss of the

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22 peptide can be expected due to the acid lability of the linker, thus slightly decreasing the yield during each N-deprotection step. Additionally, not all sequences are stable to the harsh acidic conditions used during HF cleavage and orthogona l deprotection resulting in truncation of the desired peptide. Due to these caveats several solid phase peptide synthetic methodologies have been developed that utilize milder reaction conditions and do not require special equi pment as alternative for the Boc synthesis strategy.92 O H N O OH O O O H2N HN O O O O OTFA TFAPiperidineA c i d l a b i l e P e p t i d e R e s i n L i n k e rBase-labile Temporary Protection Group Acid-labile Permanent Protection Groups Insoluble Polymeric Support Figure 2-5. The Fmoc solid-phase synthetic strategy The most common alternative to th e Boc technique has been the 9-fluroenylmethoxycarbonyl (Fmo c) strategy (Figure 2-5).93,94 The method involves using the base-labile Fmoc group for temporary N protection, acid-labile permanent protecting groups for reactive side chains, a nd acid-labile linkers. Much milder reaction conditions are used because the mechanism for removal of the temporary Fmoc protecting group is significantly different from that of the permanent protecting groups on the reactive side chains. Hence Fmoc remova l between coupling reactions does not affect side chain protection. Additiona lly, since the linker is acid labile, repeated exposure to basic solutions does not result in loss of pep tide from the resin due to premature cleavage.

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23 A possible mechanism of Fmoc removal is shown in Figure 2-6 under basic conditions with 20% piperidine in dimethylformamide (DMF).92,95 The side chain protecting groups and linker to the polymeric support are generally cl eaved together in one step with a TFA solution. Appropriate scav engers are added to prevent unwanted side reactions during cleavage and depr otection as in the Boc strategy. O O N H H O O N H NH N H H H2N O O N H -CO2 Figure 2-6. Possible mechanism for base-catal ylized removal of the N-terminal Fmoc protection group. Coupling Methods The critical step in peptid e synthesis is the condensa tion of two amino acids to create the characteristic peptide bond or amide bond. The incoming amino acid carboxy group is activated and then reacted with the free amino portion of the resin bound amino acid. The activate amino acid should be highl y reactive so that the coupling reaction proceeds as close to 100% completion as possibl e. Since the vast majority of amino acids have a chiral center at the –carbon, coupling reactions must also proceed with minimal loss of stereostructural integrity. Obtaini ng the balance between reactivity and minimal racemization is often difficult to accomp lish because the activated amino acids are

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24 generally highly reactive species with good leav ing groups that can increase the acidity of the –proton resulting in racemization through en olization of the activated amino acids.92 Figure 2-7 illustrates two possible mechanisms of racemization of activated amino acids. The two main coupling techniques used in peptide synthesis are through in situ activation of the carboxylic acid of the amino acid with a su itable activating or coupling reagent and the use of pre-activated species already purified and characterized. In our laboratory the first of the two methods is employed and a description of the most common method used for in situ activation, with carbodi imides, phosphonium or aminium salts is explained further. R2N H Act O H R1R2N H Act OH R1N H Act O H R1O N H R1O R2O N OH R2R2O R1HActEnolization OxazoloneFormation Figure 2-7. Possible mechanisms of activated amino acids racemization Carbodiimides Carbodiimide activation, until about 1985, was the most widely used coupling reagents in solid-phase synthesis with N,N-dicyclohexylcarbodiimide (DCC) and N,Ndiisopropylcarbodiimide (D IC) the reagents of choice (Figure 2-8). DCCDICNCN NCN Figure 2-8. Structures of widely used carbodiimides

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25 The carbodiimide-mediated activation of the amino acid is complex and not completely understood, but is strongly depe ndent on the solvent. In a polar solvent (DMF) activation is slow due to the formati on of several ac tive intermediates, as opposed to a nonpolar solvent (CH3Cl) with the O-acylisourea being the predominant intermediate (Figure 2-9).92 The transfer of a proton and additi on of the carboxylic acid forms the very reactive O-acylisourea intermediate which can undergo a number of different reactions. Since the first step in the mechanism is pr otonation of the carbod iimide, addition of a base prior to amino acid ac tivation should be avoided. NCN RR HOR1O NCN RR H OR1O NCN RR H O O R1O O R1R1O O N H R1O Rx OR1O N H R1O R2R2NH2R2NH2R2NH2Symetrical Anhydride Oxazolone O-acylisoureaamino attackNCN RR H O R1ON-acylureaR e a r r a n g e m e n to t h e r c a r b o x y l i c a c i d a t t a c kN c a r b o x a m i d e o r c a r b a m a t e a m i n o a c i d a t t a c k ( i n t r a m o l e c u l a r c y c l i z a t i o n ) Figure 2-9. Possible mechanisms of amide bond formation via carbodiimide activation Figure 2-9 shows possible mechanisms of carbodiimide activation leading to the formation of O-acylisour eas, N-acylurea, symmetrical anhydrides, and 5(4H)-

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26 oxazolones.92 Each of the possible intermediate is reactive, eventually leading to amide bond formation. The formation of Oxazolone in creases the possibility of racemization (Figure 2-7), thereby increasing the possibi lity of unwanted side products. Oxalone formation can be minimized by addition of a less reactive nucleophile, such as a 1-hydroxybenzotriazole (HOBt), which gene rates an active ester derivative.92 Active esters are less reactive than O-acyli soureas, but are far more stable intermediates and significantly reduce the risk of racemization (Figure 2-10). CNH N O H N O R Y N N N HO HO H N O R Y N H CN H OHOBtN CNCNH N O H N O R Y O N N NDICO H N O R Y N N N.. ++ +1,3-Diisopropyl-urea protected amino acid Y = temporary protecting groupO-acylisourea Active ester Figure 2-10. A possible mechanism of active ester formation using carbodiimides (DIC) and 1-hydroxybenzotri azoles (HOBt). Figure 2-10 illustrates a possible mechanis m of active ester formation using a carbodiimide and either one of the two co mmonly used hydroxybenzotriazoles, HOBt or 1-hydroxy-7-azabenzotriazole (HOAt, with N at position 7 of the aromatic ring).92 The

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27 esters formed from addition of either HOBt or HOAt are less reactive than O-acylisoureas of carbodiimide reducing the possib ility of side reactions. The concentration of the active ester can be increased with th e addition of one equivalent of hydroxylamine, to aid in reducing the formation of other inte rmediates, such as oxazolones, thus reducing the possibility of racemization especially when DMF is used as the solvent.92 Upon addition of the active ester to the peptide-resi n, one equivalent of a hindered base such as diisopropylethylamine (DIEA) is added to accelerate the coupling reaction.96 Onium salts NP X N N R RR R RR NC X N R RR RY Y Phosphonium Aminium Figure 2-11. General structure of phosphonium and aminium salts Phosphonium and aminium salts, referred to as “onium” salts can be used for the activation of carboxylic acid in the preparati on of amides and peptides (Figure 2-11). Figure 2-12 shows the most common onium salts used for amino acid activation in solidphase peptide synthesis. The exact mechanism of phosphonium activat ion is currently not known, but it is postulated that the mechanism proceeds through a highly reactive acyloxyphosphonium salt to form either an active ester or a symmetrical anhydride.97-102 The mechanism of aminium activation is believed to proceed through an acyloxy-guanidino intermediate that reacts immediately with the hydroxybenz otriazole base pres ent in the reaction medium. The hypothesized mechanism of amin ium salt activation is shown in Figure 2-13A. Aminium reagents can lead to N-termin al guanidino derivatives , and thus terminate

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28 chain elongation (Figure 2-13B). Due to the possibility of guanidation, pre-activation with 0.9 equivalents of aminium reagents per amino acid prior to addition to the peptide-resin is optimal to suppress the unwanted side reaction N N N O PN N NPF6N N N O PN N NPF6N N N N O PN N NPF6N N N N O PN N NPF6N N N C O N N N N N N C O N NPF6N N N C O N NBF4N N N N C O N NBF4PF6A B CD E F G H Figure 2-12. Structures of the most comm on aminium and phosphonium salts used for amino acid activation in solid-phase synt hesis as coupling reagents. A) n-[1HBenzotriazole-1-yl(dimethylamino) methylene]-N-methylmethanaminium Hexafluorophosphate N-oxide (HBTU). B) N-[(Dimethyl amino)-lH-l,2,3triazolo[4,5b]pyridino-l-ylmet hylene]-N-methylmethanaminium hexafluorphosphate N-oxide (HAT U). C) N-[1H-Benzotriazole-1yl)(dimethylamino)methylene]-N-methyl methanaminium tetrafluoroborate Noxide (TBTU). D) N-[(Dimethylami no)-lH-l,2,3-triazolo[4,5b]pyridino-lylmethylene]-N-methylmethanaminium te trafluoroborate N-oxide (TATU). E) Benzotriazol-l-yl-N-oxy-tris(dimethylamino)phosphonium Hexafluorophosphate (BOP). F) Benzotriazol-l-yl-N-oxytris(pyrrolidino)phosphonium Hexa fluorophosphate (PyBOP). G) (7Azabenzotriazol-1-yloxy)tr is(dimethylamino)phosphonium Hexafluorophosphate (AOP). H) (7-Azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium Hexafluorophosphate (PyAOP). Monitoring Procedures It is often convenient to m onitor the progression of the coupling reaction, especially during manual synthesis. It should be noted th at for short to medium length sequences (approximately 15 residues) the following methods provide a reliable means of

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29 monitoring the status of coupling reactions, however the results can be misleading for longer peptide sequences.92 O O H N R YPF6 N H N N N N N O O O H H N R Y NN OHBTUN N N O N O N H R Y O N N N O N H R Y O N N O N H R Y N N O O N N N+ +++ DIEA protected amino acid Tetramethyl-urea Y =temporary protecting groupA BO O H N R HHBTUO O H N R N N Figure 2-13. Amino acid activia tion with HBTU coupling reag ent an aminium salt. A) Possible mechanism of active-ester formation. B) Peptide chain termination due to N-termin al guanidation side reaction. The most widely used method of monito ring the coupling reaction is the Kaiser (Ninhydrin) test.103 The test provides a fast and co nvenient colorimetric test for the detection of free amines. Ninhydrin reacts with free amines to produce the dye

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30 “Ruhemann’s purple” which is read ily visible to the naked ey e (Figure 2-14). The Kaiser test can be adapted for use in both qualitative and quantitative analyses. O O O C R NH2C O Peptide H O O C R N C O Peptide HH2OO O C R N C O PeptideOH H O O C R N C O PeptideHOO NH2OH2OC R O C O Peptide+O N O O O O O O+ +Ruhemann's purple Ninhydrin Figure 2-14. Possible mechanism of Ruhemann’ s purple formation using Kaiser’s reagent to monitor the progressi on peptide synthesis. Secondary amines, such as proline, do not produce the typical colorimetric response during the Kaiser tes t; therefore alternative met hods must be employed when monitoring proline and other se condary amino acids. The chlora nil test is another rapid and convenient color test that can be us ed to detect both primary and secondary amines.92,95 Chloranil (2,3,5,6-tetrachloro-1,4-b enzoquinone) reacts with primary and secondary amines to form the green-b lue 2,3,5-trichloro-6-(2 -pyrrolidinylvinyl)[1,4]benzoquinone derivative (F igure 2-15). Since the benz oquinone derivative produces

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31 a greenish color visible to the naked eye, the chloranil test provides a rapid test for the presence of secondary amines (Figure 2-16). Cl ClCl CH O O CH N O N H Figure 2-15. Possible structure of chloranil reaction product. Chloranil reacts with the secondary amino acid proline to pr oduce the blue-green benzoquinone derivative. This colorimetric response provides a rapid method to monitor the presence of secondary amines. NinhydrinTest Positive (free primary amines present) Negative (No free amines present) ChloranilTest Positive (free 2oamines present) Negative (No free amines present) NinhydrinTest Positive (free primary amines present) Negative (No free amines present) ChloranilTest Positive (free 2oamines present) Negative (No free amines present) Figure 2-16. The two colorimetric assays used to monitor peptide synthesis. A positive response (color formation) indicates the presence of free amino-termini. Purification and Analysis Following the cleavage and orthogonal depr otection steps, the crude peptide is precipitated by addition of cold (4 C) ethyl ether. After pr ecipitation, the peptide can be filtered over a course glass frit and washed with additional cold ether to improve purity. The precipitated peptide can then either be extracted (o ften with neat acetic acid or acidic aqueous solutions) and lyophilized, or simply dried in vacuo before purification. Peptide quality is routinely analyzed by using analytical high performance reversed-phase liquid chromatography (RPHPLC) to access purity and mass spectral

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32 analysis to assure the correct molecular wei ght. The majority of crude peptides can be purified to homogeneity with relative eas e using semi-preparative HPLC in the appropriate solvent system. Analysis of the purified sample is accomp lished by either analytical HPLC in two diverse solvent systems or th in layer chromatography (TLC) in three solvent systems. Elemental and amino and analysis can also pr ovide further detail of peptide content. Additionally, data from 1H-NMR can provide reliable info rmation regarding structural integrity. Development of Peptidomimetics In 1902, Emil Fischer and Franz Hofmeister discovered that prot eins and peptides are made up of amino acids linked by amid e bonds. James D. Watson and Francis Crick (1953) about 51 years later proposed the double helical structure of DNA and postulated that the nucleotide sequence in DNA carries encoded genetic information. This discovery led to an understandi ng that it is the genetic code that determines the amino acid sequences of proteins and peptides. Since these discoveries, a large number of bioactive and na turally-occurring peptides has been identified and characterized. Peptide receptors have been identified as drug discovery targets because peptides ar e known to influence a large number of essential physiological processes. The intrinsic properties of peptides, such as poor bioavailabil ity, short biological half-lives and low receptor selectivity have limited their use as drugs. Due to these limitations, most bioactive peptides are not used as drugs. Instead they are used as starting points in the search for small “d rug-like” organic molecules that mimic the biologically active peptide, designated, peptide mimetics or peptidomimetics .

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33 General Strategies for the De velopment of Peptidomimetics Most endogenous peptides exert their e ffects through binding a nd activation of G-Protein-coupled receptors (GPCRs). The direct and structure-based design of peptidomimetics would benefit from three dime nsional (3D) structural data of complexes between the peptide and their GPCR target. Unfortunately, X-ray structures of GPCRs are limited to that of bovine rhodopsin, reported by Palczewski in 2000.14 Because of this limitation, more indirect, ligand-base d design methodologies are applied. Biologically Active Peptide A Structure-Activity Relationships alanine scan D-amino acid scan deletions, truncations Minimal and Essential Fragment B Constrained Peptide Analogues local constraints (spec. amino acids) global constraints (cyclization) secondary structures mimetics (peptide-like)Bioactive Conformation, 3D pharmacophore model C Topographical Design of Non-Peptide Scaffold Mimetics Non-Peptide Ligands Biological TestingBiophysical StudiesNMR Molecular ModelingScreening Combinatorial libraries Natural products synthetic librariesNon-Peptide LigandsHit Lead Optimization Figure 2-17. Rational strategy for the development of peptidomimetics

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34 In solution peptides are present in a nu mber of different conformations because they are very flexible molecules, and ar e dependent on the solvent used. Receptors, however, often select a minor conformer and a ssist in transforming this conformation into the one that binds and activates the receptor. Rational strategies for the design of pepti domimetics is depicted in Figure 2-17 and have been outlined by several groups.104-108 A brief discussion of each step follows: A. After identifying the primary sequence of a biologically active peptide, the structure-activity relationship (SAR) of the individual amino acids should be explored. Systematic exchange of the di fferent amino acids, for example by alanine (alanine scan), or by D-amino acids, and reduction of the peptide length, will lead to identification of a minimal fragment n eeded for recognition and activation of the receptor. B. The next step will be to synthesize conformationally co nstrained peptide analogues in order to limit the possible relative orie ntations of the key residues identified as required for recognition and activation. The e ffects of the induced constrains should be carefully analyzed using biophysical methods, such as NMR and molecular modeling. This step may provide inform ation about the receptor-bound and/or biologically active conformation, and may optimally lead to a hypothesized 3D pharmacophore model. C. In the third step, the topographic in formation obtained from 3D pharmacophore models can be used to construct a n on-peptide mimetic where the important pharmacophores have been mounted onto a ca refully selected non-peptidic scaffold or template. When discussing strategies for rational discovery (not only rational design) of peptidomimetics, then random screening shoul d be added. This includes screening of natural products, in-house s ynthetic collections, or lib raries from combinatorial chemistry. A large number of compounds that are peptidomimetics have evolved by optimization of lead structures f ound in such screening programs. The entries in Figure 2-17 should be used in an iterative manner and requires reliable biological testing me thods. However, the design of “drug-like” non-peptides

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35 from peptides remains the ultimate challenge, since it requires comprehensive understanding of the structural requir ements for recognition and activation.

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36 CHAPTER 3 STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF MONOCYCIC AGRP AT THE MOUSE MELANOCORTIN RECEPTORS Portions of the study presented in this ch apter have been prev iously published and have been reproduced in part from 1) Joseph, C. G.;Bauzo, R.M.;Xiang, Z.; Shaw, A. M., Milliard, W. J., Haskell-Luevano, C. Peptides., 2003 , 24 , 263-270 and 2) Joseph, C. G.; Wang, X. S.; Scott, J. W.; Bauzo, R.M.; Xia ng, Z.; Richards, N. G.; Haskell-Luevano, C. J. Med. Chem., 2004, in press. All peptides were de signed, synthesized, purified, and analytically characterized by Christine G. Joseph under the supervision of Dr. Carrie Haskell-Luevano. The pharmacology studies were carried out by Rayna M. Bauzo, Joseph W. Scott and Dr. Zhimin Xiang, all members of the Haskell-Luevano laboratory group. Data analysis was performed by Dr . Carrie Haskell-Luevano. Modeling was performed by Xiang S. Wang in the department of Chemistry. Introduction Two lines of evidence suggested the exis tence in the brain of an “Agouti-like” protein that would bl ock signaling at central melanocor tin receptors, MC3R and MC4R. First, in vitro pharmacology studies found that Agouti was a highly specific MC4R antagonist even though it was normally expressed only in hair follicles.60,66,109 Second, central administration of synthetic MC3R and MC4R antagonists uncovered a functional role for melanocortin antagonists in vivo , namely, the stimulation of feeding behavior.74 The agouti-related protein (AGRP) gene, was isolated in 1997 based on its homology to Agouti.67,70

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37 AGRP is a 132-amino acid protein with a signal sequence and a Cys-rich C-terminal domain. AGRP contains 10 Cys re sidues in the C-terminal domain, that participate in the formation of fi ve disulfide bridges (Figure 3-1)60,110 that are essential for its structural stability and biological function. Biochemica l studies indicate also that AGRP is very stable to thermal dena turation as well as acid degradation.111 In addition, the biophysical characterization of AGRP shows that its seco ndary structure consist of mainly random coils -sheets and a cystine knot.60,62,111,112 RCVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCRKLGTAMNPCSRThAGRP (86-132) Figure 3-1. Disulfide arrangement of the C-terminal domain of human AGRP. Cys residues are depicted in red. AGRP is a competitive antagonist of -MSH action at melanocortin receptors.111,113,114 Likewise, the C terminus of AGRP (residues 87) retains the biological activity of th e full-length protein in vitro113 as well as in vivo.115 AGRP is equally potent in inhibiting signaling at the central melanocortin receptors, MC3R and MC4R (binding affinity of human AGRP close to 1 nM for both receptors), with very little inhibition detected at the MC5R, and virtually no activity detected at MC1R.111,114,116 AGRP is expressed primarily in the hypotha lamus, adrenal medulla, and at low levels in testis, lung, and kidney.67,70 The localization pattern of human and murine AGRP is strikingly alike,70 indicating similar roles for AGRP in both species. Brain expression of AGRP mRNA is confined to neuronal ce ll bodies localized in the arcuate nucleus of the hypothalamus.117 These neurons are shown to project to hypothalamic

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38 nuclei that receive dense pro-opiomelanocortin stimulation and express the two central melanocortin receptors, MC3R and MC4R.117 The potency of AGRP action at MC3R and MC4R together with their si milar distribution pattern suggests that AGRP controls their function in vivo . The presence of AGRP in a subset of hypothalamic nuclei (i.e. , arcuate, paraventricular, dorsomedial) strongly suggests a key role for AGRP and the melanocortin system in the regulation of energy homeostasis. This conclusion is supported by multiple findings. First, central administration of AGRP is shown to mimic the effect of synthetic MC3R and MC 4R antagonists and stimulate feeding.115 In addition, AGRP is able to specifically bl ock the reduction in food intake elicited by administration of -MSH.74 Second, overexpression of AGRP in transgenic animals results in an obesity phen otype strikingly similar to that of the MC4RKO mouse,67,118 suggesting that hypothalamic AGRP is an im portant endogenous stimulator of feeding and exerts this function by inhibiting me lanocortin agonist signaling. Therefore, melanocortinergic neurons exert a tonic inhi bition on feeding beha vior and metabolism and this tonic inhibition is relaxed following AGRP antagoni sm at MC4R resulting in stimulation of food intake and energy storage. Elongation of Yc[CRFFNAFC]Y There has been great intere st in the MC4 receptor since it has been reported as being important in the regulation of feeding.40,74 The MC4R knockout mice exhibit hyperphagia and become obese.40 Both the MC3 and MC4 recep tors are expressed in the brain, but the MC3 receptor i nvolvement in the regulation of food intake is unclear. The MC3 receptor knockout mice exhibit increased fat mass but are not significantly

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39 overweight,36,37 indicating that the MC3 and MC4 recep tors serve different roles in the regulation of energy homeostasis. Development of ligands that can discriminate between the MC3 and MC4 receptors will be of great therapeutic benefit in the treatment of obesity and its related diseases. Therefore, it is important to identify the molecular determinants of the receptor that enables MC3/MC4 receptor discrimination as well as the key amino acid residue(s) of the ligands that determines selectivity. Tota et al. showed that the AGRP(1 09-118) decapeptide Y-c[CRFFNAFC]Y binds to both the hMC3R (IC50 = 1.9 M) and hMC4R (IC50 = 57 nM) with reduced binding affinity of 420-fold and 15-fold respectivel y, compared to the C-terminal domain of AGRP.68 However, the decapeptide only antagonizes the hMC4R (Ki = 785 nM).68 This was later confirmed in our laboratory w ith AGRP(109-118) binding to the mouse MC1R, MC3R, MC4R and MC5R and was only an antagonist at the mMC4R with Ki = 158 nM.88 Pharmacological characterization of th e decapeptide at the mouse melanocortin receptors resulted in MC1R agonist activity88 which was a very interesting observation, since AGRP has no binding or f unctional activity at the MC1R.67,113 Based upon the above information and bindi ng data from the truncation of agouti and AGRP into monocyclic, bicyc lic and tricyclic derivatives,119 it was hypothesized that extension beyond the core decapeptide of AGRP (109-118), at either (or both) the Nand C-terminus should re-establish MC3R f unctional activity. The main idea was to determine the minimal active fragment for MC3R antagonism as well as the use of a monocyclic template to reduce the number of disulfide bridges, hence getting rid of synthetic complexity.

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40 Results Chemical Synthesis and Characterization The peptides reported herein were synthesized using standard fluorenylmethyloxycarbonyl (Fmoc)93,94 chemistry with a manual reaction vessel (Peptides International, Louisville, KY). Th e peptides were purified to homogeneity using semi-preparative reversed-phase high pressure liquid chromatography (RP-HPLC). The purity of these peptides was assessed by mass spectrometry and analytical RP-HPLC in two diverse solvent systems (Table 3-1). Table 3-1. Analytical data of the monocyclic AGRP peptides 1-8 Peptide Sequence HPLC k’ (system 1) HPLC k’ (system 2) % purity m/z (M, calcd) m/z (M + 1, expt) 1 Yc[CRFFNAFC]Y-NH2 5.9 12.2 > 99 1330.5 1331.0 2 DPAATAYc[CRFFNAFC]Y-NH2 6.2 11.5 > 96 1857.1 1857.7 3 Yc[CRFFNAFC]YARKL-NH2 6.2 11.3 > 99 1799.1 1799.6 4 TAYc[CRFFNAFC]YARKL-NH26.3 11.4 > 99 1971.3 1972.1 5 DP AATAYc[CRFFNAFC]YARKL 6.5 12.6 > 99 2326.7 2327.3 6 GQQVPAADPAATAYc[CRFFNAFC]YARKL-NH2 6.6 11.8 > 98 2977.4 2978.3 7 DPAATAYc[CRFFNAFC]YAR-NH2 6.4 11.7 > 97 2084.3 2085.4 8 TAYc[CRFFNAFC]YAR-NH2 6.1 10.9 > 98 1730.0 1729.6 HPLC k’ = [(peptide retention time – solvent re tention 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 ac id/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 was determined by HPLC at a wavelength of 214 nm. Biological Evaluation We have designed, synthesi zed and pharmacologically characterized eight hAGRP derivatives at the mouse melanocortin MC1R , MC3R, MC4R and MC5R receptors. The ability of peptides 1-8 to competitively antagonize the mMC3 and mMC4 receptors and to stimulate the mMC1R and mMC5R was exam ined using the functional assay described

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41 in Chapter 7, and the results are summarized in Table 3-2. None of the peptides examined in this study possessed mMC5R agoni st or antagonistic properties. Table 3-2. Pharmacology of the monocyclic AGRP peptides 1-8 at the mouse melanocortin receptors Antagonist pA2 Agonist EC50(nM) Peptide Sequence mMC3RmMC4R mMC1R hAGRP(86-132) 8.9 0.29.4 0.1 NA 1 Yc[CRFFNAFC]Y-NH2 NA 6.4 0.5 4400 860 2 DPAATAYc[CRFFNAFC]Y-NH2 NA NA 19600 1900 3 Yc[CRFFNAFC]YARKL-NH2 6.2 0.96.9 0.6 2200 260 4 TAYc[CRFFNAFC]YARKL-NH2 6.6 0.67.3 0.9 820 170 5 DPAATAYc[CRFFNAFC]YARKL 6.8 0.37.5 0.2 600 240 6 GQQVPAADPAATAYc[CRFFNAFC]YARKL-NH2 6.3 0.76.9 0.6 890 220 7 DPAATAYc[CRFFNAFC]YAR-NH2 6.5 0.47.1 0.7 1800 490 8 TAYc[CRFFNAFC]YAR-NH2 6.1 0.76.6 0.8 950 220 NA denotes neither agonist nor antagonist pharmacology was observed at or up to 100 M concentrations. pA2=-Log Ki. The Ki values were used to determine the fold differences of the antagonist va lues relative to the hAGRP (109-118) decapeptide. These peptides did not possess agonist or antagonist pharmacology at the mMC5R (data not shown). Mouse melanocortin-1 receptor All the peptides ( 1-8 ) possessed mMC1R full agonist activity that ranged from 600 nM to 19 M EC50 agonist values (Table 3-2). The most potent mMC1R agonist of the peptides studied herein is analogue 5 which is 7-fold more potent than 1, hAGRP (109-118). Mouse melanocortin-3 receptor The peptides in this library were tested for antagonist activity (Table 3-2) and binding affinity at the mMC3R (Table 33). The competitive binding studies were performed with radiolabeled I125-NDP-MSH and I125-hAGRP (86-132) and the results are shown in Table 3-3. Peptide 2, like peptide 1, lacked the ability to stimulate a functional response at the mMC3R. The AGRP decapeptide (109-118) 1 was able to competitively displace radiolabeled NDP-MSH but only 42 % specific binding was observed at 10 M concentrations with radiolabel AGRP (86-132) at the mMC3R. Elongation of 1 at the

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42Table 3-3. Binding results fo r AGRP derivatives using 125I-NDP-MSH and 125I-AGRP radioactive labels in the assays to determine inhibitory binding concentration (IC50) at 50% the maximal response [125I-NDP-MSH] IC50 (nM) [125I-AGRP(86-132)] IC50 (nM) Peptide Sequence mMC3R Fold diff mMC4R Fold diff mMC3R mMC4R Fold diff NDP-MSH Ac-Ser-Ty r-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 0.5 0.05 0.9 0.4 ND ND AGRP hAGRP (86-132) 2 0.3 2 0.6 2 1 3 0.3 1 Yc[CRFFNAFC]Y-NH2 8800 3000 1 300 30 1 42% 1000 60 1 2 DPAATAYc[CRFFNAFC]Y-NH2 0 % 19 % 24 % 10 % 3 Yc[CRFFNAFC]YARKL-NH2 1000 20 -9 100 10 -3 1200 500 200 10 -5 4 TAYc[CRFFNAFC]YARKL-NH2 800 50 -12 70 10 -4 500 4 100 1o -10 5 DPAATAYc[CRFFNAFC]YARKL 3500 300 -2.5 200 10 1 1500 200 200 10 -5 6 GQQVPAADPAATAYc[CRFFNAFC]YARKL-NH2 3300 1310 -2.5 200 30 1 1100 200 70 40 -14 7 DPAATAYc[CRFFNAFC]YAR-NH2 8400 3000 1 200 20 1 7300 600 400 100 -2.5 8 TAYc[CRFFNAFC]YAR-NH2 1600 100 -5.5 50 1 -6 1400 300 100 3 -10 % indicates the percent total specific binding of the peptide at up to10 M. ND indicates no binding activity was observed.

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43 N-terminal by the addition of six amino acids provided peptide 2 [hAGRP (103-118)], with reduced binding affinity at the mMC3R. At 10 M concentrations, peptide 2 was able to competitively displace 125I -AGRP by 19% but not the 125INDP-MSH label at the mMC3R. Extension of peptide 1 [hAGRP (109-118)] at the C-terminal by four amino acids, peptide 3 [hAGRP (109-122)], possessed high nM antagonist activity at the mMC3R and was able to competitively displaced radiolabeled NDP-MSH and hAGRP (86-132) with M IC50 values at the MC3R. Peptide 4 [hAGRP (107-122], peptide 5 [hAGRP (103-122), and Peptide 6 [hAGRP (96-122)] were extensions of peptide 3 at the N-terminal. Peptide 5 is the most potent mMC3R antagonist (pA2 = 6.8, Ki = 158 nM) and possessed mMC3R binding IC50 values of 1500 – 3500 nM (Table 3-3). C-terminal removal of Lysine and Leucine of peptides 4 and 5 resulted in peptides 7 and 8. Comparison of peptides 4 and 8 resulted in a 3-fold differen ce in antagonist potency at the mMC3R, which is within the inherent 3fold experimental error (Table 3-2). Both peptides did not result in significant diffe rences in ligand binding affinities at the mMC3R using either radiolabeled peptides . Extension of the Cterminal region of hAGRP (109-122) by thirteen amino acids (endogenous AGRP Cys residues in this region have been substituted with Ala to exclude the formation of multiple disulfide bridges), peptide 6 , resulted in equipotent binding a nd functional activity compared to analogue 3. Mouse melanocortin-4 receptor The hAGRP (109-118) peptide 1 (Yc-[CRFFNAFC]Y-NH2) possessed antagonist activity (Ki = 398 nM) at the mMC4R that was 995fold less potent than C-terminal AGRP(86-132). Peptide 1 was also able to competitively displace radiolabeled NDP-

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44 MSH and radiolabeled AGRP at this receptor with 150to 330-fold reduced affinity compared to C-terminal AGRP. Elongation of peptide 1 [hAGRP (109-118)] at the Nterminal by the addition of six amino acids, peptide 2 [hAGRP (103-118)], resulted in complete lost of functional activity at the mMC4R. At 10 M concentrations, peptide 2 was able to competitively displace 125INDP-MSH and 125I -AGRP by 10-19 % at the mMC4R (Table 3-3). Extension of hAGRP (1 09-118) at the C-terminal by four amino acids, peptide 3 [hAGRP (109-122)], possesse d antagonist activity (Ki =126 nM) at the mMC4R and was able to competitively displaced radiolabeled NDP-MSH and hAGRP(86-132) with 100 nM and 200 nM IC50 values respectively, at the MC4R. Peptide 4 [hAGRP(107-122], peptide 5 [hAGRP(103-122), and Peptide 6 [hAGRP (96122)] are extension of peptide 3 at the N-terminal. Peptide 5 is the most potent mMC4R antagonist (pA2 = 7.5, Ki = 32 nM, Table 3-2 and Figur e 3-3), and possessed mMC4R binding IC50 values of 200 nM with both radiolabel ed ligands (Table 3-3). C-terminal removal of Lysine and Leucine of peptides 4 and 5 resulted in peptides 7 and 8. Comparison of peptides 4 and 8 resulted in a 5-fold change in MC4R antagonist potency (Table 3-2). Both peptides did not result in significant differences in ligand binding affinities at the mMC4R using either radiolabeled peptides. Extension of the C-terminal region of hAGRP(109-122) by thirteen ami no acids (endogenous AGRP Cys residues in this region have been substituted with Ala to exclude the formation of multiple disulfide bridges), peptide 6 , resulted in equipotent binding a nd functional activity compared to analogue 3.

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45 Discussion Extension of monocyclic AGRP(109-118) at th e Cand N-terminal identified two 14 amino acid monocyclic derivatives, peptides 3 and 8, as the minimal fragments with antagonist activity at the mMC3R (Tab le 3-2). However, all the monocyclic AGRP peptide derivatives with binding affinity or antagonist activity had a slightly higher affinity (10to 35-fold) and were more potent antagonist for the mMC4R over the mMC3R (Tables 3-2 and 3-3) but none were hi ghly selective for either receptor. It has been proposed that the Arg-Ph e-Phe triplet residues conserve d in both agouti protein and AGRP are necessary for melanocortin receptor antagonism 62,68,88,109,112,120,121 because when any one of these residues were replaced with alanine it resulted in loss of binding affinity and was not an antagonist of the MC4R.68,109 Previous reports indicated that the C-terminal 87-132 residues of hAGRP posse ss equipotent affinity for both MC3R and MC4R.67,113 Although the derivatives ( 3-8 ) are antagonists and bind to the mMC4 and mMC4 receptors, they do so with reduced functional activities and binding affinity compared to AGRP(86-132). Thus, it can be hypo thesized that other structural features missing in our monocyclic derivatives and present in AGRP(86-132) are required for high affinity and potency at the mMC3R and mMC4R. One factor that may be contributing to this difference is the overall tertiary struct ure due to the five disulfide bridges present in the endogenous AGRP ligand, while the derivatives contains only one disulfide bridge. However, there may be other s ubtle structural features still needed to be identified. Two radiolabeled peptides were utilized to determine if a putative different binding “epitope” was observable at the mMC3 and mMC4 receptors for agonist (NDP-MSH) versus antagonist [AGRP(86-132)]. 125I-NDP-MSH, a linear 13 amino acid agonist

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46 peptide (Table 3-3) and 125I-AGRP(86-132), a 46 amino acid antagonist peptide containing five disulfide bridges (Figur e 3-1) were used for competitive binding displacement studies. The two radiolabeled ligan ds resulted in iden tical binding affinity at the mMC3 and mMC4 receptors within e xperimental error with each of the AGRP derivatives, 1-8 (Table 3-3). Comparison of th e radiolabeled NDP-MSH versus hAGRP(86-132) to competitively non-iodi nated hAGRP(86-132) at the mMC3 and mMC4 receptors also resulted in equipotent binding IC50 values. Based on the results it can be suggested that radiolabeled NDP-MSH could be used to competitively displace AGRP derivatives at the mMC3 and mMC4 recep tors since it costs much less to prepare NDP-MSH that AGRP(86-132) both in the term s of money and time. Since there was no difference in binding affinity with these two radiolabels, it suggests that there is a common “binding epitope” at the mMC3 a nd mMC4 receptors for both NDP-MSH and hAGRP(86-132). The above observation suppor ts the hypothesis generated by several laboratories,62,67,68,109,112,113,119,122-124 but does not exclude the concept of melanocortin antagonist such as AGRP(86-132) or melanoc ortin agonists having additional distinct putative ligand-receptor interactions. Unexpectedly, as postulated, extension of the core hAGRP(109-118) monocyclic decapeptide past the addition of four amino acids at the N-terminus did not result in a significant increase in mMC3R antagonism. Th ere was only up to a 12-fold increase in antagonist potency gained by the addition of resi dues at both the Nand C-terminal of the core hAGRP(109-118) monocyclic decapeptide at the mMC4R. It was proposed that the inclusion of the QQ hAGRP(97-98) amino acids would putatively increase melanocortin receptor potency which was determined base d upon the high-resolution NMR structure of

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47 the AGRP C-terminus.62,112,123 Modification of the QQ AGRP residues to alanines in the AGRP template resulted in equipotent bindi ng affinities at th e human MC3 and MC4 receptors compared to AGRP.68 In this study, peptide 6 possessing the hAGRP(96-122) monocyclic sequence with the QQ hAGRP(97-98 ) residues did not increase antagonist activity compared to peptide 5 that lacked the N-termin al hAGRP(96-102) amino acids. However, there was a 14-fold in crease in binding affinity of 6 at the mMC4R using the I125-hAGRP(86-132) radiolabel, as compared with the core hAGRP(109-118) decapeptide 1, although the endogenous AGRP Cys residues in this region were replaced with Ala residues. Peptide 6 had equipotent binding affinity with both radiolabeled ligands compared to 5. Because the derivatives in this study contained one disulfide bridge, the results obtained in no way precludes the hypot hesis that increased melanocortin potency will be gained by the addition of the AGRP QQ residues important for the threedimensional AGRP structure and activity in analogues that possess more than one disulfide bridge.62,112,123 Stereochemical Inversion of Arg111-Phe112-Phe113 Positions Careful comparison of Agouti and AGRP C-terminal sequences reveals, however, the presence of a conserved Arg-Phe-Phe motif that resembles the -MSH pharmacophore His-Phe-Arg-Trp.68 A loop of eight residues flanked by two Cys residues and including the Arg-Phe-Phe triplet (AGRP residues 110 and Agouti residues 115, respectively) is shown to be critic al for both Agouti and AGRP antagonism at melanocortin receptors.68 Furthermore, Alanine (Ala) scanning mutagenesis studies indicate that the Arg-Phe-Phe motif is the mo st critical in determin ing antagonist activity (IC50 = 0.5 0.1 nM for AGRP binding to MC4R whereas IC50 for the three Ala mutants

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48 are: 67 46 nM for Arg111Ala, 61 35 nM for Phe112Ala, and 25 13 nM for Phe113Ala, respectively).68 The octapeptide loop of the antagonist is therefore proposed to mimic the conformation of -MSH and interact with th e receptor through a similar mechanism.68 This model would thus imply that the agonist and antagonist occupy the same binding site on the receptor. An alte rnative model suggests that the antagonist attaches itself to a different receptor site and blocks ligand binding through an allosteric mechanism. In support of this model it was re cently shown that the extracellular loops 2 and 3 of the MC4R are critical sites for an tagonist (AGRP) binding but had little effect on agonist ( -MSH) binding.116 Based on the above informati on we designed a library of seven peptides using the mo st potent monocyclic analogue 5 from the elongation study and performed stereochemical invers ion of the Arg-Phe-Phe residues. This study was undertaken to determine wh ether stereochemical conversion of ArgPhe-Phe amino acid residues of monocyclic hAGRP(103-122) would result in enhanced potency and/or MC3/MC4 receptor selectivit y. Homology modeling of selective peptide analogues in the mMC4R was performed to locate the regions and residues of the mMC4R and the ligand that may be responsib le for agonist and antagonist functional activity. Results Chemical Synthesis and Characterization The peptides reported herein were synthesized using standard fluorenylmethyloxycarbonyl (Fmoc)93,94 chemistry with an Advanced Chemtech 440MOS automated synthesizer (Advanced Ch emTech, Louisville, KY). The peptides were purified to homogeneity using semi-prepa rative reversed-phase high pressure liquid

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49 chromatography (RP-HPLC). The purity of these peptides was assessed by mass spectrometry, and analytical RP-HPLC in tw o diverse solvent systems (Table 3-4). Table 3-4. Analytical data of the monocyclic AGRP peptides with stereochemical conversion Peptide Sequence HPLC k’ (system 1) HPLC k’ (system 2) % purity m/z (M, calcd) m/z (M + 1, expt) 5 DPAATAYc[C-Arg-Phe-Phe-NAFC]YARKL 6.5 12.6 > 99 2326.7 2327.3 9 DPAATAYc[C-DArg111-Phe-Phe-NAFC]YARKL 6.6 11.2 > 96 2326.7 2328.0 10 DPAATAYc[C-Arg-DPhe112-Phe-NAFC]YARKL 6.5 11.2 > 99 2326.7 2327.6 11 DPAATAYc[C-Arg-Phe-DPhe113-NAFC]YARKL 6.6 11.5 > 97 2326.7 2326.9 12 DPAATAYc[C-DArg111-DPhe112-Phe-NAFC]YARKL 6.3 11.0 > 99 2326.7 2326.6 13 DPAATAYc[C-DArg111-Phe-DPhe113-NAFC]YARKL 5.9 10.9 > 98 2326.7 2328.1 14 DPAATAYc[C-Arg-DPhe112-DPhe113-NAFC]YARKL 5.9 10.5 > 99 2326.7 2326.1 15 DPAATAYc[C-DArg111-DPhe112-DPhe113-NAFC]YARKL 6.0 12.0 > 98 2326.7 2327.0 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/water a nd a gradient to 90% methanol over 35 min). An analytical Vydac C18 colu mn (Vydac 218TP104) was used with a flow rate of 1.5 m L/min. The peptide purity was determined by RP-HPLC at a wavelength of 214 nm . Pharmacology We have designed, synthesi zed and pharmacologically characterized eight hAGRP derivatives at the mouse melanocortin MC 1, MC3, MC4 and MC5 receptors. The ability of peptides 9-15 to competitively antagonize the mMC3 and mMC4 receptors and to stimulate the mMC1R and mMC3-5R was examin ed using the functional assay described in chapter seven and the results are summarized in Table 3-5. Mouse melanocortin-1 and -5 receptors All the derivatives containing one or mo re stereoisomer of Arg-Phe-Phe were agonist at the mMC1 and mMC5 receptors. The lead peptide 5 was a high nanamolar agonist at the mMC1R (EC50 = 960 nM) with no agonist or antagonist activity at the mMC5R.The D-Arg111 (peptide 9 ) was a weak agonist at the mMC1R and mMC5R. The

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50Table 3-5. Functional activity of monocyclic hAGRP(103-122) pept ide analogues with stereochemical conversion at the mouse melanocortin receptors AGONIST EC50 (nM) ANTAGONIST pA2 Peptide Sequence mMC1R mMC3R mMC4R mMC5R mMC3R mMC4R MTII Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 0.02 0.01 0.2 0.04 0.04 0.005 0.2 0.1 -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe -Arg-Trp-Gly-Lys-Pro-Val-NH2 0.3 0.04 0.5 0.06 1.9 0.2 1.12 0.36 NDP-MSH Ac-Ser-Ty r-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 0.02 0.008 0.09 0.02 0.1 0.2 0.06 0.01 hAGRP C-terminal fragment (86-132) NA NA NA NA 8.9 0.2 9.4 0.1 5 DPAATAYc[C-Arg-Phe-Phe-NAFC]YARKL 960 240 NA NA NA 6.2 0.2 6.9 0.3 9 DPAATAYc[C-DArg111-Phe-Phe-NAFC]YARKL 6330 3880 70%@100000 37700 8560 18300 5350 10 DPAATAYc[C-Arg-DPhe112-Phe-NAFC]YARKL 93 36 50% @100000 75% @ 8290 1410 280 6.1 0.9 6.6 1.1 11 DPAATAYc[C-Arg-Phe-DPhe113-NAFC]YARKL 120 45 13700 1310 3700 1140 2220 920 12 DPAATAYc[C-DArg111-DPhe112-Phe-NAFC]YARKL 1750 570 5510 60 8300 1970 4600 1700 13 DPAATAYc[C-DArg111-Phe-DPhe113-NAFC]YARKL 5270 990 70% @100000 8330 840 19800 9060 14 DPAATAYc[C-Arg-DPhe112-DPhe113-NAFC]YARKL 64 17 63300 13700 6240 1000 11400 2430 15 DPAATAYc[C-DArg111-DPhe112-DPhe113-NAFC]YARKL 3750 95 22600 7490 21500 3190 13200 4230 Errors are the standard error of the mean from at least three independent experiments. The pA2 antagonist value was determined by Schild analysis (pA2 = -log Ki). NA denotes no agonist activity.

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51 D-Phe112 peptide 10, the D-Phe113 peptide 11 , the D-Arg111-D-Phe112 peptide 12 , the D-Arg111-D-Phe113 peptide 13 , the D-Phe112-D-Phe113 peptide 14 , and the D-Arg111D-Phe112-D-Phe113 peptide 15 were all agonist at the mMC1R and mMC5R. Peptide 14 was a potent and selective ligand for the mMC1R (EC50 = 64 nM). Mouse melanocortin-3 and -4 receptors Intracellular cAMP accumulation functi onal assay revealed that analogue 5 is a weaker antagonist at the mMC3R (Ki=708 nM) and mMC4R (Ki = 120 nM) when compared to hAGRP (86-132). The D-Arg111 (peptide 9 ) had no antagonist activity at the mMC3 and mMC4 receptors, but had high M agonist activity at the mMC4R and became a partial agonist at the mMC3R with maximum efficacy of 70% at 100 M relative to MT-II (Table 35). The D-Phe112 peptide 10 maintained antagonist activity at the mMC3R and mMC4R but were also a partia l agonist at these tw o receptors. The DPhe113 peptide 11 , the D-Arg111-D-Phe112 peptide 12 , the D-Arg111-D-Phe113 peptide 13 , the D-Phe112-D-Phe113 peptide 14 , and the D-Arg111-D-Phe112-D-Phe113 peptide 15 showed no antagonist bi oactivity bu t were all M agonist at the mMC3 and mMC4 receptors with peptide 13 being a partial agoni st at the mMC3R. Modeling into the mMC4R Peptide 5 containing the endogenous Arg-PhePhe (111-113) triplet, peptide 9 with a D-Arg111, and peptide 11 with a D-Phe113 were docked into the mMC4R to identify residues important for agonist and antagonist activities (Figure 3-2). The hAGRP Arg111 amino acid of peptide 5 was observed to be interacting with the mMC4R in a negatively charged pocket formed by the melanocortin receptor residues Glu92 (TM2), Asp114 (TM3) and Asp118 (TM3). The D-Arg111 of peptide 9 was also observed as interacting

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52 with this negatively charged pocket. However, a strong interaction was also seen between the hAGRP D-Arg111 amino acid of 9 and the mMC4R Asn115(TM3) that was not seen with Arg111 of peptide 5 (Figures 3-2B and 3-4). Peptide 5 Phe112 and Phe113 were observed to be interacting with an aromatic-hydrophobic pocket consisting of the mMC4R Phe176 (TM4), Phe193 (TM5), Phe253 (TM6) and Phe254 (TM6) (Figures 32C and 3-4). Although Phe112 and D-Phe113 of 11 were observed to be interacting with the same aromatic-hydrophobic pocket as 5 , the D-Phe113 of 11 had a much stronger interaction with Phe176 (TM4) co mpared to Phe113 of peptide 5 (Figure 3-2C). Discussion The goal of the stereochemical inversion study was to identify a lead template of the Arg-Phe-Phe (111-113) residues common to both melanocortin endogenous agonist agouti and AGRP that might result in increase potency and selectivity at the mMC3 and mMC4 receptors. The Arg-Phe-Phe (111-113) of hAGRP has been shown to be critical for high affinity binding and activity.68 AGRP is an antagonist at the MC3 and MC4 receptors and identification of key amino acid residues that are necessary for receptor recognition would help in the design of mo re potent and selective ligands for these receptors. Monocyclic hAGRP (103-122) cont aining 20 amino acid residues and one disulfide bridge has been shown previously to be an antagonist of the MC3R and MC4R.125 However, it was not significantly selec tive for either receptor (Table 3-5). Monocyclic hAGRP (103-122) containing one disulfide bridge between Cys residues at position 110 and 117 (hAGRP numbering) was used as a lead in a novel series of analogues. The Cys residues at positions 105, 108 and 119 were replaced with Ala to eliminate any possibility of mo re than one bridge being formed and the stereochemistry of Arg-Phe-Phe (111-113) were inverted individually as well as two or more residues at

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53 the same time. When the Arg-Phe-Phe (111-113) residues of hAGRP were replaced with alanine there were a reduction of bindi ng affinity at both the hMC3R and hMC4R,68 indicating that the Arg-Phe-Phe residues were necessary for binding to these receptors. Although an alanine scan was done for Arg-PhePhe, a D-amino acid scan of these three residues were never tried before to determ ine the importance of stereochemistry. The D-enantiomer may render the ligand into a unique conformation and thus promote differences in potency and selectivity at the melanocortin receptors. Whenever the Arg111 and/or Phe113 were repla ced with their D-enan tiomer in the series there were a complete lost of antagonist ac tivity at the mMC3 and MC4 receptors and the analogues became either agonist or partial a gonist at the mMC1R, and mMC3-5R (Table 3-5). The functional results implies that the endogenous Lenantiomer of Arg111 and Phe113 are necessary and appears to be involved in maintaining mMC3R and mMC4R antagonism. Peptide 10 , with D-Phe112 remained an e quipotent antagonist at the mMC3 and mMC4 receptors compared peptide 5 supporting this obser vation. Intriguingly, peptide 10 became a full agonist at the mMC5R, a partial agonist at the mMC3 and mMC4R and is a 10-fold more potent agonist at the mMC1R. Due to the difference in pharmacological results between peptide 5 and its derivatives ( 9-15 ) it can be hypothesized that the change in stereochem istry of Arg-Phe-Phe (111-113) may have placed all the analogues in a bi oactive conformation that was favorable for agonist or partial agonist activity at the mMC3R or mMC4R and full agonist activity at the mMC5R. The stereochemical inversion of th e Arg-Phe-Phe (111-113) triplet resulted in the discovery of a new agonist template for the melanocortin receptors. The study resulted in peptide 14 containing D-Phe112D-Phe113 (EC50 = 64 nM) as a potent

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54 mMC1R agonist with selectivity of 989-fold over the mMC3R, 98-fold over the mMC4R and 178-fold over the mMC5R. Generally, al l the analogues were more potent at the mMC1R. DR111 Asn115mMC4R Peptide 9 DF113Phe176Peptide 11 mMC4R mMC4R Peptide 5 A BC DR111 Asn115 DR111 Asn115mMC4R mMC4R Peptide 9 Peptide 9 DF113Phe176 DF113Phe176Peptide 11 mMC4R mMC4R mMC4R mMC4R Peptide 5 Peptide 5 A BC Figure 3-2. Model of the monocyc lic hAGRP(103-122)-mMC4R complex. The amino acids of the receptor involved in inte ractions with the ligands are colored by atom type: C, white; O, red; and N, blue. A. Ribbon diagram of the monocyclic peptide 5-mMC4R model, vi sualized using CAChe V5.0. Peptide 5 ligand in white and TM helical do main (1-7) of mMC4R in green. The Arg111 of the ligand in yellow and Phe112 and Phe113 in red side chains of the important residues involved in the hydrophobic or aromatic interactions are highlighted and labeled (Lamino acid of ligand in yellow and D-amino acid in purple). B. Ribbon diagram of the monocyclic peptide 9 -mMC4R model. The side chains of the impor tant residues involved in the ionic interactions are highlighted a nd labeled (DArg111 of peptide 9 in purple and mMC4R Asn115 in yellow). C. Ribbon diagram of the monocyclic peptide 11 -mMC4R model. The side chains of th e important residues involved in the hydrophobic interactions are highlighted and labeled (DPhe113 of peptide 11 in purple and mMC4R Phe176 in green)

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55 Modeling into the mMC4R Agouti-related protein (AGRP) has been show n to be involved in the regulation of food intake via the MC4 receptor and ster eochemical inversion of hAGRP(103-122) converted an mMC4R antagonist to mMC4R ag onist (Table 3-5). Based on these data, homology molecular modeling was performed to provide insights into the molecular basis of these derivatives as antagonist and agonist. To loca te the regions and residues that may be responsible for agonist and antagonist functional, peptide 5 an mMC4R antagonist, peptide 9 a high M mMC4R agonist and peptide 11 a 10-fold more potent mMC4R agonist (Table3-5, Figur e 3-3) was modeled into the mMC4R receptor (Figure 3-2). The docking of peptides 5 , 9 , and 11 in the mMC4R showed the positively charged Arg111 of 5 and 11 and the D-Arg111 of 9 as interacting with negatively charged residues in TM2 and TM3 of the mMC4R. The ionic interacti on between Arg111 of 5 and 11 with the mMC4 receptor Glu92 (TM2), Asp114 (TM3) and Asp118 (TM3) appears to be important for agonist a nd antagonist binding. Melanocortin-4 receptor mutagenesis data122,126 support this observation because point mutation of the mMC4R Glu92Lys and Asp118Lys resulted in reduced binding affinity of hAGRP(86-132) and had no antagonists activity.122 Replacement of Arg111 in Cterminal AGRP with alanine resulted in approximately 130-fold decr eased binding affinity for the hMC4R68 supporting the above observation. However, with peptide 9 , the additional interaction of D-Arg111 with the ionic Asn115 (TM3) of mMC4R (Figure 32B) not seen with Arg111 of peptides 5 and 11 may be a key factor in converti ng the peptides with D-Arg111 from mMC4R antagonist to agonists.

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56 mMC4R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.25MT-II 10,000 nM 5+ MT-II 1000 nM 5 + MT-II 100 nM 5 + MT-II 10 nM 5 + MT-II 5 Log Concentration MT-II [M]-Galactosidase Normalized to Protein & Forskolin mMC4R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.25MT-II 9 Log Concentration MT-II [M]-Galactosidase Normalized to Protein & Forskolin mMC4R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.25MT-II 11 Log Concentration MT-II [M]-Galactosidase Normalized to Protein & ForskolinA B C Figure 3-3. Illustration of f unctional activity of the anal ogues used for modeling into mMC4R. Graph A : peptide 5 , hAGRP(103-122) possessing antagonist activity at the mMC4R. Graph B : Peptide 9 , hAGRP(103-122, DArg111) possessing slight agonist activity at the mMC4R. Graph C : Peptide 11 , hAGRP(103-122, DPhe113) possessing fu ll agonist activity at the mMC4R Both Phe113 ( 5 and 9 ) and D-Phe113 ( 11 ) were observed as interacting in a hydrophobic binding pocket consisting of Phe 176 (TM4), Phe193 (TM5), Phe253 (TM6) and Phe254 (TM6) of the mMC4R. This in teracting between the ligands and the hydrophobic binding pocket may be involved in li gand-receptor interaction because both agonist ( 9 and 11 ) and antagonist ( 5 ) are interacting with these same residues. The importance of these residues for molecular recognition, binding and functional antagonism of AGRP(86-132) and the decap eptide AGRP(109-118) containing the ArgPhe-Phe (111-113) triplet was shown with mutagenesis studies of mMC4R.122 When Phe113 of AGRP was mutated it resulted in decreased binding affi nity ant the hMC4R,68 indicating the importance of th is residue for antagonist binding. Point mutation of Phe176

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57 of mMC4R resulted in no f unctional antagonist activity with was shown to be important AGRP(86-132) and AGRP(109-118), indicating its importance fo r functional antagonism The conversion of the mMC4R antagonist, peptide 5 to a mMC4R agonist, peptide 11 may be due to the strong hydrophobic interaction between D-Phe113 of 11 and mMC4R Phe176 (TM 4) that is lacking with Phe113 of 5 . A C D K N Y W T P Y R M S R S S CI P Y F CI I K K F T R R Q S A Y Y Y I I I I P P D V V A N N N C C C C M M F F S S H H P Q FF FF Y I I I I I H P A A W V V V V C C F T T T T K M N T Q R R R P A A I I K H M NFF M M M M H Y V A S V T I I I V A S S S D Y I IFV V V V T T S S I I I I I C C A A W R R M H Y Y Q A A A F F F I I I I I I T T Y R DD DV V V V S S S S S S S S C CNN N Q A D D T T T T I I I V V V V S S S S S N N N M M D A A A C F F Y P H K K I I I I A V V V V V V N S S F F T P Q Y H S S N SH Y S S NH2HOOC G E L L E G G L E L E L L L L L L L L L L G G G G G L L L L L L L G L G G L L L L L L L L L LEG L L L L L E E E G G G G GL L LN115 E92 D114 D118 F176 F193 F253 F254 Figure 3-4. Schematic Diagram of the mouse Melanocortin-4 Receptor . The putative transmembrane spanning domains and loop regions are based upon the 2.8 structure of rhodopsin with slight extension of the TM5 domain and the addition of two amino acids as part of the second extracellular loop. The horizontal gray lines represent the approximate membrane boundaries. Red circles with white text indicate the amino acids involved in the putative hAGRP(103-122)-mMC4R interactions. Substitution of the third and fourth tran smembrane (TM) domains of the MC4R with the MC1R TM domains resulted in lost of AGRP(110-117) binding affinity and decreased AGRP(87-132) binding affinity,127 providing further support that the Arg-PhePhe (111-1113) was interacting with these TM domain of the MC4R. There was also a

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58 decrease or lost in functional activity for these two ligands due to this substitution indicating that the third and fourth TM is involved in binding and function. Hence the interaction of our analog ues with Asn115 (TM3) and Phe176 (TM4) may explain the pharmacological differences observed. Summary The above elongation study identified two 14-amino acid sequences, TAYc[CRFFNAFC]YAR-NH2 and Yc[CRFFNAFC]YARKL-NH2 as the minimal fragments require for mMC3R antagonism, su pporting our hypothesis. However, none of the ligands were selective antagonists for either the mMC3R or mMC4R and they were all mMC1R agonists. The stereochemical study identified a novel agonist template DPAATAYc[CRFFNAFC]YARKL with D stereo chemistry at the Arg-Phe-Phe (111113) positions for the MC1R and MC3-5R . Modeling of these analogues into the mMC4R indicates that the interaction of D-Arg111 with mMC4R Asn115 (TM3) and DPhe113 with mMC4R Phe176 (TM4) may be responsible for changing an mMC4R antagonist into an agonist. It will be important in the future to de termine whether this determination is true by mutagenesis studies. .

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59 CHAPTER 4 TRUNCATION STUDIES OF GAMMA2-MELANOCYTE-STIMULATING HORMONE All peptides were designed, synthesized, purified and analytically characterized by Christine G. Joseph under the supervisi on of Dr. Carrie Haskell-Luevano. The pharmacology studies in human embryonic kidne y (HEK-393) cells stably expressing the melanocortin receptors were carried out by Jo seph W. Scott and Nickolas Sorensen, both members of the Haskell-Luevano laboratory group. Data analysis was performed by Dr. Carrie Haskell-Luevano. Introduction Prior to 1935, the only approach to the identification of a lead drug was through random screening. This involved the testi ng of all compounds in a bioassay without regard to their structures. The classes of materials screened were mainly synthetic chemicals and natural products from plants , microorganisms and marine life. Random screening was and still is the lead disc overy method of choice when nothing is known about the receptor target. Because of the high cost and the la rge number of manpower hours associated with random screening, in the la te 1970s a nonrandom screening (also called called targeted or focused screening) approach was adapted. With focused screening, compounds possessing a vague resemblance to weakly active compounds are tested selectively. None of the above approaches to le ad discovery involves a major rational component. The lead is found through screeni ng techniques. However, it is possible to

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60 design a compound having a particul ar activity through rational approaches. The first step of a rational approach is to identify the cause for a disease state such as the physiological and biochemical systems involved. Once the rele vant biochemical system is identified, initial lead compounds need to be identified th at could be the natura l receptor ligand or enzyme substrates. Once a lead has been id entified, the key structur al features required for activity has to be identified. In the case of a peptide, the information regarding ligand receptor interactions are obtained through experiments such as an alanine scan (replacement of each amino acid sy stematically with alanine) to determine the specific side chains involved in binding and functi onal activity; D-amino acid scan (replacement of each amino acid systematically with its D-enantiomer) to determine the importance of stereochemistry; and truncation studies (rem oval of individual amino acids from the Cand N-terminal) to determine the minimal fragments needed to retain activity and binding, as well as potency equal to that of the lead peptide.105,106,128,129 The knowledge gained from these studies is then used in designing new ligands with hopefully improved potency and selectivity for a receptor syst em. Structure-activity relationship studies (SAR) are developed around this information to provide insight into the types of interactions that occur in the formation of the ligand-receptor complex; such as the favorable and unfavorable changes of am ino acids and ligand backbone resulting in receptor stimulation or inhibition. The main obj ective of SAR studies is to aid in the design of ligands, with specific function, fo r a given receptor or receptor system. 2-MSH SAR Studies Gamma2-melanocyte-stimulating hormone 2-MSH) is a 12-amino acid peptide that is derived from the N-terminal fragment of POMC27 and contains the His-Phe-Arg-

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61 Trp motif common to all melanocortin endogenous agonist ligands. The five melanocortin receptor subtypes (MC1-5R) are about 42-67% identical to each other, yet 2-MSH preferentially activates the hMC3R.27 The MC3R knockout mice were found not to be hyperphagic or significantly overweight, but they have increased adipose tissues and increased feeding efficiency.36,37 This pattern suggests that while the MC4R regulates food intake and energy expenditure, the MC3R regulates energy homeostasis and separating of nutrients into fat.37,130 Hence, structure-activity relationship (SAR) studies of 2-MSH should be helpful in identifying key amino acid residues that allows it to be more selective for the MC3R over the other MC1, MC4 and MC5 receptor subtypes. Alanine and D-amino Acid Scans As mentioned above, two types of struct ure-activity strategi es used for characterizing a native peptide are alan ine and D-amino acid scans. The systematic replacement of the side chains of individua l amino acids with a methyl group (alanine scan) is performed to determine the importance of these side chains for interaction with the receptor. Alanine scan of 2-MSH (Tyr1-Val2-Met3-Gly4-His5-Phe6-Arg7-Trp8-Asp9Arg10-Phe11-Gly12-OH) revealed that the la st four amino acids (Asp9-Arg10-Phe11-Gly12) in the C-terminal region were not important for biological activity and selectivity at the human MC3-5 receptors cloned into CHO cells. 131 However, Met3, His5, Phe6, Arg7 and Trp8 were needed for binding affinity a nd agonist activity at these receptors.131 This same group also performed a D-amino acid scan to provide insights into the stereochemical requirements of each amino aci d residue for peptide-receptor interaction. The D-amino acid scan identified that the aromatic residues Tyr1, Phe6, Trp8 and Phe11 and the basic Arg10 residue as being important for human MC3R selectivity over the

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62 MC4 and MC5 subtypes.132 The study also identified DTrp8 as being extremely important for hMC3R potency and selectivity.132 Truncation of 2-MSH The above information provided us with so me insights about the important residues needed for 2-MSH binding affinity and functional activity, so the next step was to determine the minimal sequence needed to produce a pharmacological response. Because of the importance of the melanocortin system, pa rticularly the MC3 and MC4 receptors in energy homeostasis and obesity, therapeutic ag ents for these receptor subtypes are being pursued. One of the classical ra tional design approaches involv es truncation of the parent ligand to identify smaller and more manageable fragments as lead for further studies, which may eventually lead to the deve lopment of nonpeptides. Using the above information a library of twenty-eight 2-MSH analogues were designed, synthesized, and characterized at the mouse melanocortin r eceptors with truncation at the Cand N-terminal. The objective of this st udy was to perform truncation of 2-MSH to determine the minimal active peptide sequence and the amino acid residues necessary for molecular recognition. Results Peptide synthesis. The peptides reported herein we re synthesized using standard fluorenylmethyloxycarbonyl (Fmoc)93,94 chemistry and a parallel synthesis strategy on an automated synthesizer (Advanced ChemTech 440MOS, Louisville, KY). The peptides were purified to homogeneity using semi-prepa rative reversed-phase high-pressure liquid chromatography (RP-HPLC). The purities of these peptides were assessed by mass spectrometry, analytical RP-HPLC in two diverse solvent systems (Table 4-1).

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63 Table 4-1. Analytical data of 2-MSH analogues synthesized in this study Peptide Sequence HPLC k’ (system 1) HPLC k’ (system 2) % purity m/z (M, calcd) m/z (M + 1, expt) 16 Tyr-ValMet -Gly-His-Phe-ArgTrp -Asp-ArgPhe-Gly-OH 5.3 9.9 >99 1570.8 1571.4 17 Tyr-Val-Met-Gly-His-Phe-ArgDTrp8 -AspArg-Phe-Gly-OH 5.0 9.6 >96 1570.8 1570.9 18 Tyr-ValNle3 -Gly-His-Phe-Arg-Trp-Asp-ArgPhe-Gly-OH 5.5 10.4 >97 1552.7 1552.2 19 Tyr-ValNle3 -Gly-His-Phe-ArgDTrp8 -AspArg-Phe-Gly-OH 5.3 10.1 >97 1552.7 1554.4 20 Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-ArgPhe-OH (1-11) 6.0 10.3 >98 1513.7 1513.7 21 Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-ArgOH (1-10) 4.5 8.5 >98 1366.6 1366.0 22 Tyr-Val-Met-Gly-His-Phe-Arg-TrpOH (1-8) 6.0 10.0 >99 1095.3 1095.0 23 Tyr-Val-Met-Gly-His-Phe-Arg-OH (1-7) 3.5 7.0 >99 909.1 908.7 24 Tyr-Val-Met-Gly-His-Phe-OH (1-6) 4.4 8.5 >98 752.9 753.1 25 Tyr-Val-Met-Gly-His-OH (1-5) 2.3 4.3 >96 605.7 606.2 26 Tyr-Val-Met-Gly-OH (1 -4) 2.7 4.9 >98 468.6 468.9 27 Tyr-Val-Met-OH (1-3) 2.9 5.5 >99 411.5 413.3 28 Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-PheGly-OH (2-12) 5.1 10.6 >97 1407.6 1408.0 29 Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-GlyOH (3-12) 5.0 9.3 >98 1308.5 1308.3 30 Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (412) 4.8 8.8 >98 1177.1 1177.4 31 His-Phe-Arg-Trp-Asp-Arg-Phe-GlyOH (5-12) 4.8 9.0 >98 1120.2 1120.2 32 Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (6-12) 4.9 9.0 >98 983.1 983.7 33 Arg-Trp-Asp-Arg-Phe-Gly-OH (7 -12) 3.9 7.2 >98 835.9 836.5 34 Trp-Asp-Arg-Phe-Gly-OH (8-12) 3.7 6.7 >99 679.7 680.5 35 Asp-Arg-Phe-Gly-OH (9-12) 2.3 3.9 >99 493.5 494.4 36 Arg-Phe-Gly-OH (10-12) 2.8 3.4 >99 378.4 379.2 37 Tyr-Met-His-Phe-Arg-Trp-Phe-OH (1 ,3,5-8,11)6.4 10.7 >98 1086.2 1086.9 38 Met-Gly-His-Phe-Arg-Trp-OH (3-8) 5.5 9.0 >99 833.0 832.9 39 His-Phe-Arg-Trp-Asp-Arg-Phe-OH (5-11) 4.9 9.4 >98 1063.2 1063.4 40 Phe-Arg-Trp-Asp-Arg-Phe-OH (6-11) 5.0 9.4 >99 926.0 926.5 41 Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-OH (3-11) 5.7 9.7 >99 1251.4 1251.2 42 Asp-Arg-Phe-OH(9-11) 3.3 4.5 >99 436.4 437.1 43 His-Phe-Arg-Trp-OH (5-8) 4.5 8.8 >97 644.7 645.0 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 ac id/water and a gradient to 90% methanol over 35 min). An analytical Vydac C18 colu mn (Vydac 218TP104) was used with a flow rate of 1.5 m L/min. The peptide purity was determined by HPLC at a wavelength of 214 nm.

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64Table 4-2. Functional activity of the 2-MSH analogues at the mous e melanocortin receptors EC50 (nM) Peptide Sequence mMC1R Fold diff. mMC3R Fold diff. mMC4R Fold diff mMC5R Fold diff. 16 ( 2-MSH) Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH 628 175 1 40 6 1 416 63 1 39 8 1 17 Tyr-Val-Met-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OH 117 44 -5 90 46 2 5600 2300 13 7 2 -6 18 Tyr-ValNle3-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH 417 115 -1.5 1200 500 30 33000 16400 79 163 102 4 19 Tyr-ValNle3-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OH 432 268 -1.5 56 25 1 6900 3300 17 455 284 12 20 Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe -OH (1-11) 1500 400 2 155 43 4 1100 300 3 56 21 1.6 21 Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-OH (1-10) 249 60 -2.5 36 9 1 659 197 1.6 31 16 1 22 Tyr-Val-Met-Gly-His-Phe-Arg-Trp--OH (1-8) 2900 1000 5 2800 1000 70 4200 600 10 614 155 16 23 Tyr-Val-Met-Gly-His-Phe-Arg--OH (1-7) 2810 6700 4.5 36600 2400 partial agonist 915 >100000 11700 2500 300 24 Tyr-Val-Met-Gly-His-Phe--OH (1-6) >100000 >100000 >100000 >100000 25 Tyr-Val-Met-Gly-His-OH (1-5) >100000 >100000 >100000 26800 3400 687 26 Tyr-Val-Met-Gly-OH (1-4) >100000 >100000 >100000 >100000 27 Tyr-Val-Met--OH (1-3) >100000 >100000 >100000 >100000 28 Val-Met-Gly-His-Phe-Arg-Trp-As p-Arg-Phe-Gly-OH (2-12) 1300 500 2 400 250 10 830 348 2 82 24 2 29 Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (3-12) 1600 500 2.5 259 67 6 1200 500 3 94 15 partial agonist 2 30 Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (4-12) 7900 2700 13 760 239 19 2700 600 6.5 294 152 7.5 31 His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (5-12) 20100 5900 32 2700 100 68 8400 2700 20 759 286 19 32 Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (6-12) >100000 >100000 47400 17700 114 13500 7200 partial agonist 346 33 Arg-Trp-Asp-Arg-Phe-Gly-OH (7-12) >100000 >100000 >100000 >100000 34 Trp-Asp-Arg-Phe-Gly-OH (8-12) >100000 >100000 >100000 >100000 35 Asp-Arg-Phe-Gly-OH (9-12) >100000 >100000 >100000 >100000 36 Arg-Phe-Gly-OH (10-12) >100000 >100000 >100000 >100000 37 Tyr-Met-His-Phe-Arg-Trp-Phe-OH (1,3,5-8,11) 67100 0 107 10400 2300 260 14000 3700 partial agonist 34 1100 200 28 38 Met-Gly-His-Phe-Arg-Trp-OH (3-8) 14500 2100 23 22400 5200 560 41900 11800 101 797 211 20 39 His-Phe-Arg-Trp-Asp-Arg-Phe-OH (5-11) 44800 6100 71 6600 700 165 64400 19800 partial agonist 155 1900 620 49 40 Phe-Arg-Trp-Asp-Arg-Phe-OH (6-11) 3200 840 5 20400 3600 510 34000 10650 82 6700 1700 172 41 Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-OH (3-11) 1800 300 3 366 89 9 2400 800 6 101 23 3 42 Asp-Arg-Phe-OH (9-11) >100000 >100000 >100000 >100000 43 His-Phe-Arg-Trp-OH (5-8) >100000 >100000 >100000 48700 800 partial agonist 1249 Indicated errors represent the standard error of the mean, determined from at least four independent experiments. >100000 indic ate that no agonist activity was observed at up to 100 M. Partial agonists means that the stimulatory response was less than 100%

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65 Biological evaluation. Table 4-2 compares the cAMP stimulation ( -Galactosidase assay) of the twenty-eight 2-MSH analogs ( 2-28 ) with that of the native peptide, 1 , at the cloned mouse MC1, MC3, MC4 and MC5 receptors. Mouse melanocortin-1 receptor. The MC1R is expressed in melanocytes and is involved in human skin pigmentation21,25,133 and coat coloration.133 The native 2-MSH peptide, Tyr-Val Met-Gly-His-Ph e-Arg-Trp-Asp-Ar g-Phe-Gly-OH ( 16 ), possesses a 628 nM EC50 at the mMC1R herein. In this study at the mMC1R, the DTrp analogue 17 resulted in 5-fold increased agonist activity (EC50 = 117 nM) at the mMC1R compared to 16. Replacement of Met3 with Nle3 resulted in equipotent mMC1R potency compared with 16. Analogue 4 containing both Nle3 and DTrp8 resulted in equipotent agonist activity (EC50 = 432 nM) compared with 16. Agonist activity was maintained at the mMC1R when the first five residues (Gly-P he-Arg-Asp-Trp) were removed from the C-terminal (peptides 20-23 ). Peptides 20 (Gly removed) and 21 (Gly-Phe removed, Figure 4-1) both had equipotent (within experimental error) agonist activity compared to 16 at the mMC1R. Peptides 22 and 23 were 5and 45-fold less potent than 16, respectively, at the mMC1R. Further amino acid removal from the C-terminal (peptides 24-27 ) resulted in loss of stimulatory activity at up to 100 M at the mMC1R. The first four amino acid residues (Tyr-Val-Met-Gly) can be removed from the N-termin al without loss of agonist activity (peptides 28-31 ). Peptides 28 and 29 had equipotent (within experimental error) agonist activity. Peptides 30 and 31 resulted in reduced potency ranging from 13to 32fold, respectively. Peptide 32-36 with further N-terminal residue removal resulted in loss of stimulatory activity at up to 100 M. Peptide 37 containing all the residues that were shown to be important for binding and func tional activity at the human melanocortin

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66 receptors,131,132 resulted in M agonist activity and 107-fold decreased potency at the mMC1R. When both Cand N-terminal truncation was performed at the same time (peptides 38-43 ) there was generally a reduction a pot ency ranging from 3to 71-fold. Interestingly, peptide 43 containing the His-Phe-ArgTrp pharmacophore common to all the endogenous melanocortin agonists resulted in complete loss of agonist activity at up to 100 M at the mMC1R. mMC1R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.252-MSH 21 Log peptide Concentration [M]-galactosidase normalized to protein & forskolin Figure 4-1. Illustration of 2-MSH analogue 21 an equipotent agonist compare to 2-MSH at the mouse MC1R Mouse melanocortin-3 receptor. The MC3R is believed to be involved in metabolism and energy homeostasis a nd is expressed both centrally and peripherally.22,26,36,37 The lead peptide 2-MSH, Tyr-Val Met-Gl y-His-Phe-Arg-Trp-AspArg-Phe-Gly-OH ( 1 ), has been previously reported to possess a 5.9 and 1.0 nM agonist EC50 at the hMC3R131,132 and possess a 40 nM EC50 at the mMC3R herein that may be due to species differences. It has been prev iously reported that s ubstitution at the Trp8 position with DTrp resulted in a potent and selective agonist for the hMC3R.132 In this

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67 study at the mMC3R the DTrp analogue 17 , resulted in equipotent (EC50 = 90 nM, within experimental error) agonist activity at the mMC3R compared to 16 . Replacement of Met3 with Nle resulted in 30-fold decreased mMC3R potency compared with 16 . Analogue 19 containing both Nle3 and DTrp8 resulted in equipotent agonist activity (EC50 = 56 nM) compared with 16 . Agonist activity was maintained at the mMC3R when the first five residues (Gly-Phe-Arg-Asp-Trp) were removed from the C-terminal (peptides 20-23 ). Peptide 20 , with Gly removed resulted in a 4-fold decreased in potency. Interestingly, peptide 21 , with both Gly and Phe removed from th e C-terminal is an equipotent agonist (EC50 = 36 nM) compared to 16 (Figure 4-2). Peptide 22 was 70-fold less potent than 16 and peptide 23 was a high M partial agonist with 915-fold decreased potency at the mMC3R compared to 16 . Further amino acid removal fr om the C-terminal (peptides 2427 ) resulted in loss of stimula tory activity at up to 100 M at the mMC3R. The first four amino acid residues (Tyr-Val-Met-Gly) can be removed from the N-te rminal without loss of agonist activity but with reduced pote ncy ranging from 6to 68-fold (peptides 28-31 ). Peptide 32-36 resulted in loss of stimul atory activity at up to 100 M. Peptide 37 containing all the residues th at were shown to be importa nt for binding and functional activity at the human melanocortin receptors,131,132 resulted in M agonist activity and 260-fold decreased potency at the mMC3R. Wh en both Cand N-terminal truncation was performed at the same time (peptides 38-43 ) there was generally a reduction in potency ranging from 9to 560-fold. Interestingly, peptide 43 containing the His-Phe-Arg-Trp pharmacophore common to all the endogenous melanocortin agonists resulted in complete loss of agonist activity at up to 100 M at the mMC3R.

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68 mMC3R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.252-MSH 21 Log Peptide Concentration [M]-galactosidase normalized to protein & forskolin Figure 4-2. Illustration of 2-MSH analogue 21 an equipotent (within experimental error) agonist compare to 2-MSH at the mouse MC3R Mouse melanocortin-4 receptor. The MC4R expressed in the brain has been identified as participating in food intake74 and disruption of this gene leads to obesity in mice40 as well as observed obesity in huma ns due to polymorphisms of the MC4R.76,7880,134 The lead peptide 2-MSH, Tyr-Val Met-Gly-HisPhe-Arg-Trp-Asp-Arg-Phe-GlyOH ( 1 ), has been previously reported to possess a 260 and 55 nM agonist EC50 at the hMC4R131,132 and possess a 416 nM EC50 at the mMC4R herein. It has been previously reported that substitution at the Trp8 position with DTrp resulted in a equipotent (within experimental error) agonist for the hMC4R.132 In this study at the mMC4R the DTrp analogue 17 , showed a 13-fold decrease in agonist activity at the mMC4R compared to 16 . Replacement of Met3 with Nle resulted in 79-fold decreased mMC4R potency compared with 16 . Analogue 19 containing both Nle3 and DTrp8 resulted in a 17-fold decreased agonist activity (EC50 = 6.9 M) compared with 16 . Agonist activity was maintained at the mMC3R when the first four residues (Gly-Phe-Arg-Asp) were removed

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69 from the C-terminal (peptides 20-22 ). Peptide 20 (Gly removed) and 21 (Gly-Phe removed, Figure 4-3), both had equipotent (w ithin experimental e rror) agonist activity compared to 16 at the mMC4R. Further amino acid removal from the C-terminal (peptides 23-27 ) resulted in loss of stimu latory activity up to 100 M at the mMC4R. The first five amino acid residues (Tyr-Val-M et-Gly-His) can be removed from the Nterminal without loss of agoni st activity at the mMC4R ( 28-32 ). Peptides 28 and 29 had equipotent agonist activity whereas, peptides 30-32 resulted in reduced potency ranging from 6to 114-fold. Peptide 33-36 with further N-terminal residue removal resulted in loss of stimulatory activity at up to 100 M. Peptide 37 containing all the residues that were shown to be important for binding and functional activity at the human melanocortin receptors,131,132 resulted in M partial agonist activity and 34-fold decreased potency at the mMC4R. When both Cand N-terminal truncation was performed at the same time (peptides 38-43 ) there was generally reduced potency ranging from 6to 155-fold. Interestingly, peptide 43 containing the His-Phe-Arg-Trp pharmacophore common to all the endogenous melanocortin agonists resulted in complete loss of agonist activity at up to 100 M at the mMC4R. Mouse melanocortin-5 receptor. The MC5R expressed in a variety of tissues has been implicated as participating in exocrine gland function.24,43,135 The lead peptide 2-MSH, Tyr-Val Met-Gly-His-PheArg-Trp-Asp-Ar g-Phe-Gly-OH ( 16), has been previously reported to possess a 490 and 200 nM agonist EC50 at the hMC5R131,132 and possess a 39 nM EC50 at the mMC5R herein that may be due to species difference. It has been previously reported th at substitution at the Trp8 position with DTrp resulted in a 6-fold increased agonist for the hMC5R.132 In this study at the mM C4R the DTrp analogue

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70 17 , showed a 5-fold increase in agonist activity at the mMC5R compared to 16 . Replacement of Met3 with Nle resulted in slightly reduced mMC5R potency compared with 16 . Analogue 19 containing both Nle3 and DTrp8 resulted in a 12-fold decreased agonist activity (EC50 = 455 nM) compared with 16 . Agonist activity was maintained at the mMC3R when the first five residues (G ly-Phe-Arg-Asp-Trp) were removed from the C-terminal (peptides 20-23 ). Peptide 20 (Gly removed) and 21 (Gly-Phe removed, Figure 4-4), both had equipotent (within experiment al error) agonist activity compared to 16 at the mMC5R. Further amino acid removal from the C-terminal (peptides 24-27 ) resulted in loss of stimulatory activity at up to 100 M at the mMC5R with the exception of 25 . Peptide 25 , (Tyr-Val-Met-Gly-His-OH) retained full M agonist activity at the mMC5R, but with 687-fold reduced potency. The first five amino acid residues (Tyr-Val-Met-GlyHis) can be removed from the N-terminal w ithout loss of agonist activity at the mMC4R ( 28-32 ). Peptides 28 and 29 had equipotent agonist activity with 29 being a partial agonist. Peptides 30-32 resulted in reduced potency ranging from 8to 346-fold with 32 being a M partial agonist. Peptide 33-36 with further N-terminal residue removal resulted in loss of stimul atory activity at up to 100 M. Peptide 37 containing all the residues which were shown to be important for binding and functional activity at the human melanocortin receptors,131,132 resulted in M full agonist activity and 28-fold decreased potency at the mMC5R. When both Cand N-terminal truncation was performed at the same time (peptides 38-43 ) there was generally reduced potency ranging from 3to 1249-fold. Interestingly, peptide 43 containing the His-Phe-Arg-Trp pharmacophore common to all the endogenous melanocortin agonists resulted in high M partial agonism at the mMC5R.

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71 mMC4R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.252-MSH 21 Log Peptide Concentration [M]-galactosidase normalized to protein & forskolin Figure 4-3. Illustration of 2-MSH analogue 21 an equipotent (within experimental error) agonist compare to 2-MSH at the mouse MC4R mMC5R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.252-MSH 21 Log Peptide Concentration[M]-galactosidase normalized to protein & forskolin Figure 4-4. Illustration of 2-MSH analogue 21 an equipotent (within experimental error) agonist compare to 2-MSH at the mouse MC5R

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72 Discussion To date, there is there is no data in the li terature that involves truncation studies on -MSH that is pharmacologically characte rize at the melanocortin receptors. The characterization of the native ligand agonist may provide insi ght into the mechanism of interaction with the receptor. Many peptide agonists are usually divided into a “message” (pharmacophore) region a nd an “address” region.105-107,128 The “address” region is responsible for binding of the pe ptide into the receptor, wher eas the “message” region is responsible for changing the conformation of the receptor, thus initiating G-protein interactions.105-107,128 Analysis of the native peptide to define the regi ons of binding and signal transduction is accomplished by a study of structur e-activity relationships. Among the structure-activity experiments utilize for characterization of the native ligand agonist are truncation, alanine and D-am ino acid scans. In this study truncation was performed on 2-MSH by sequential removal of amino acid residues from th e Nand C-terminal to obtain information on both the bi nding and the message regions. Modification of Met3 and Trp8 Side Chain. The lead peptide 2-MSH, 16 is shown to be an equipotent agonist at both the mMC3 and MC5 receptors with mMC3R selectivity of 16-fold over mMC1R and 10-fold over mMC4R. However, at the human receptors it was shown that 2-MSH is a selective MC3 receptor agonist with selectivity of approximately 45-fold over the hMC4R and 85-fold over the hMC5R.131,132 The differences in functiona l activity between human and mouse may be attributed to specie difference or bioassay measurement with Grieco et al. using a cAMP flashplate assay with Chinese Hampster Ovary (CHO) cells stably expressing the human melanocortin receptors131,132 and in our laboratory cAMP Response

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73 Element (CRE)/ -galactosidase reporter gene as say with Human Embryonic Kidney (HEK-293) cells stably expressing the mouse melanocortin receptors. Tryptophan8 was shown in D-amino acid scan to be critical for 2-MSH activity at the human melanocortin receptors.132 Previous studies have shown that replacement of Met with Nle resulted in an analogue136 NDP-MSH that is more potent than -MSH at stimulating adenylate cyclase on amphibian melanophores.137 Unlike Met, Nle is resistant to oxidation.138 Substitution of the Trp8 side chain ( 2-MSH numbering) with DTrp ( 17 ) in the lead peptide 16 resulted in an mMC5R selective agonist (EC50 = 7 nM) with selectivity of 17-fold over mMC1R, 13fold over the mMC3R and 800-fold over mMC4R (Figure 4-5). This analogue tested at the human melanocortin receptors by Grieco et al. was a selective hMC3R agonist with selectivity of 303-fold over the hMC4R and 248-fold over hMC5R.132 Substitution of Met3 with Nle ( 18 ) is also an mMC5R selective agonist (EC50 = 163 nM) but with a 23-fold reduced potency at this receptor and decreased selectivity over the other three receptor subtypes (Figure 4-6). Substitution with Nle3 in a C-terminal amidated analogue show ed slightly increased potency at the hMC3R but also had a reduction in selectiv ity over the hMC4 and hMC5R compared to the native 2-MSH peptide.139 Interestingly, replacement of both Met3 and Trp8 at the same time, peptide 19 , resulted in a slightly selective mMC3R agonist with selectivity of 8-fold over the mMC1 and mMC5R and 123-fo ld over the mMC4R. This analogue ( 19 ) when compared to the native 2-MSH peptide ( 16) is equipotent at the mMC3R (within experimental error), has slight ly increased ligand potency at the mMC1R and reduction in ligand potency at the mMC4 and mMC5R. Substitution with Nle3 and DNal(2’)8 in a C-terminal amidated analogue resulted in d ecreased potency at the hMC3R and hMC4R but

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74 was a selective hMC5R agonist with increased potency at this receptor compared to the native 2-MSH peptide and the Nle32-MSH-NH2 analogue.139 These data suggest that having the indole ring present at position-8 is more important than stereochemistry or addition of bulky aromatic groups at the MC3 receptor for agonist activit y because the DTrp8, peptide 19 analogue is equipotent to the native 2-MSH peptide at this receptor and the DNal(2’)8 decreased potency.139 However, further structure activity studies with substitution at the Trp8 position in 2-MSH is required to substa ntiate this observation. 0 1000 2000 3000 4000 5000 6000 mMC5RmMC3RmMC1RmMC4R17: Tyr-Val-Met-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OHEC50(nM) 7 nM 13-fold 17-fold 800-fold 0 1000 2000 3000 4000 5000 6000 mMC5RmMC3RmMC1RmMC4R17: Tyr-Val-Met-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OHEC50(nM) 7 nM 13-fold 17-fold 800-fold Figure 4-5. Illustration of Pe ptide 17 with mMC5R selectivity over the other mouse melanocortin receptors C-terminal Truncation. C-terminal truncation revealed that Phe11 and Gly12 were not necessary for maintaining agonist activity at al l four receptors tested. Peptide 21 with both these residues missing has equipotent agonist activity (within experimental error) at all four receptor. However this analogue has reduced selectivity for the mMC3 and mMC5R over the mMC1R and a slight increased in selectiv ity over the mMC4R. The data suggest that

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75 these two residues are not involved in dis tinguishing selectivity for the mMC3R or mMC5R and can be removed without losing potency at these receptors. Truncation identified Trp8 as being required for mMC4R agonist activity. 16 17 18 19 mMC5R mMC3R mMC1R mMC4R 0 5000 10000 15000 20000 25000 30000 35000EC50(nM)16 : Tyr-ValMet3-Gly-His-Phe-ArgTrp8-Asp-Arg-Phe-Gly-OH 17 : Tyr-ValMet3-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OH 18 : Tyr-ValNle3-Gly-His-Phe-ArgTrp8-Asp-Arg-Phe-Gly-OH 19 : Tyr-ValNle3-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OH 16 17 18 19 mMC5R mMC3R mMC1R mMC4R 0 5000 10000 15000 20000 25000 30000 35000EC50(nM)16 : Tyr-ValMet3-Gly-His-Phe-ArgTrp8-Asp-Arg-Phe-Gly-OH 17 : Tyr-ValMet3-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OH 18 : Tyr-ValNle3-Gly-His-Phe-ArgTrp8-Asp-Arg-Phe-Gly-OH 19 : Tyr-ValNle3-Gly-His-Phe-ArgDTrp8-Asp-Arg-Phe-Gly-OH Figure 4-6. Comparison of 2-MSH versus analogues with substitution at the 3 and 8 position in the sequence at the mouse melanocortin receptors N-terminal Truncation. N-terminal truncation of residues at position 1 to 5 revealed analogues ( 28-32) that are all slightly selective for the mM C5R over the mMC1, mMC3 and mMC4R. Nterminal truncation also revealed that His5 is necessary for mMC1 and mMC3R agonist activity and Phe6 is required for maintaining agonist ac tivity at all four receptor subtypes. Amidation and Acetylatio n of His-Phe-Arg-Trp The complete lost of agonist activity at the mMC1R, mMC3R and mMC4R and partial agonist activity at the mMC5R by Hi s-Phe-Arg-Trp-OH was unexpected since this

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76 tetrapeptide sequence is common to all me lanocortin endogenous agoni st and is believed to be responsible for lig and selectivity and stimul ation at the melanocortin receptors.53,56,140 To understand why, three analogues we re synthesized with or without amidation at the carboxy terminus and with or with acetylation at the amino terminus (Table 4-3). When the tetrapeptide were only acetylated as in peptide 44 , there was still no agonist activity at the mMC1, mMC3 and mM C4 receptors. However, it became a full agonist at the mMC5R with a 12-fold increase in potency compared to 43 . Amidation of the carboxy terminus as in peptide 46 resulted in the return of full agonist activity at the mMC1, mMC4, and mMC5 receptors and part ial agonist activity at the mMC3R when compared to peptide 43 . Peptide 45 with both carboxy terminus amidation and amino terminus acetylation resulted in full agonist ac tivity at all four receptors and was 761-fold more potent at the MC5R compared to 43 . Table 4-3. Functional activity of the am idated and acetylated His-Phe-Arg-Trp tetrapeptide analogues at the mouse melanocortin receptors EC50 (nM) Peptide ID Sequence mMC1R mMC3R mMC4R mMC5R 2-MSH NH2-YVMG HFRW DRFG-OH 628 175 40 6 416 63 39 8 -MSH Ac-SYSME HFRW GKPV-NH2 0.7 0.2 3 0.9 4 1 2 0.5 43 NH2-HFRW-OH >100000 >100000 >100000 48700 800 partial agonist 44 Ac-HFRW-OH >100000 >100000 >100000 4100 60 45 Ac-HFRW-NH 2 3500 300 3100 1300 1300 300 64 5 46 NH2-HFRW-NH 2 6800 400 2900 30 partial agonist 3300 700 120 22 The indicated errors represent the standard error of the mean determined from at least four independent experiments. This increased potency of peptide 45 may be attributed to increased ligand–receptor interactions or as pr eviously reported, the increase may be due to increase enzymatic

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77 stability.141,142 The melanocortin receptor agonist -MSH is a 13-amino-acid linear peptide that is posttranslati onally processed to include the N-terminal acetyl and Cterminal amide moieties.141,143 A study of -MSH and desacetyl-MSH (without Nterminal acetylation) showed that -MSH was more potent at the melanocortin receptors.144 In our study, the acetyla ted and amidated peptide 45 was more potent at all the receptors than the desacetyl-peptide 46, suggesting that the amino terminus acetylation is functionally significant. This is supported by previous repo rts of melanocortin peptides with modification at the N-terminus resulting in increase or decrease potencies depending on the modification made.145-151 Summary Characterization showed 2-MSH as an equipotent agonist at both the mMC3R and mMC5R with only 16-fold and 10-fold selectivity over the mMC1R and mMC4R, respectively. Replacement of Met with Nle a nd Trp with DTrp resulted in an analogue with slight selectivity for the mMC3R that was 8-fold over the mMC1R and mMC5R and was 123-fold selective over the mMC4R. Tr uncation revealed that the residues at positions 11 and 12 can be deleted from the seque nce without lost of potency at all four melanocortin receptors tested (Figure 4-7) identifying 2-MSH(1-10), peptide 21 , as the shortest sequence without lost of activity at the mouse receptors. The study identified the His at position 5 and the Arg at position 7 as necessary for agonist activity at the mMC3R. The His-Phe-Arg-Trp sequence co mmon to all melanocortin endogenous agonist needed to be amidated at the C-te rminus to have activity at the mMC1R and mMC3-5R and potency at these receptors are improved when acetylated at the N-terminus and amidated at the C-terminus.

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78 Tyr1 Val2 Met3 Gly4 His5 Phe6 Arg7 Trp8 Asp9 Arg10 Phe11 Gly12 OH Truncation possible without loss of potency Removal results in reduced potency Minimal fragment with pharmacological response Minimal fragment with pharmacological response Tyr1 Val2 Met3 Gly4 His5 Phe6 Arg7 Trp8 Asp9 Arg10 Phe11 Gly12 OH Truncation possible without loss of potency Removal results in reduced potency Minimal fragment with pharmacological response Minimal fragment with pharmacological response Figure 4-7. Illustration of 2-MSH truncation study result s at the mouse melanocortin receptors

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79 CHAPTER 5 STRUCTURE-ACTIVITY STUDIES OF UREA PEPTIDOMIMETICS AT THE MOUSE MELANOCORTIN RECEPTORS Portions of the study presented in this chapter have been pr eviously published: Joseph, C. G.;Bauzo, R.M.;Xiang, Z.; Haskell-Luevano, C. Bioorg. Med. Chem. Lett., 2003 , 13 , 2079-2082. All compounds were design ed, synthesized, purified, and analytically characterized by Christine G. Joseph under the supervision of Dr. Carrie Haskell-Luevano. The pharmacology studies we re carried out by Rayna Bauzo and Dr. Zhimin Xiang, both members of the HaskellLuevano laboratory group. Data analysis was performed by Dr. Carrie Haskell-Luevano. NMR data was obtained by Christine G. Joseph at the Advanced Magnetic Res onance Imaging and Spectroscopy (AMRIS) facility in the McKnight Brain Institute of the University of Florida with guidance from Mr. Jim Rocca. Introduction Peptides are an attractive starting poin t for drug discovery because they are essential to virtually every bi ochemical process. Unfortunatel y, the attributes of potency and specificity exhibited by peptides are c ounter-balanced by short biological half-lives and low oral bioavailability.107 One major goal of modern medicinal chemistry is to find rational approaches for systematically tran sforming the information provided in natural peptide ligands into low molecular weight nonpeptide molecules that bind to the target receptor. Chemical structures designed to c onvert the information contained in peptides into small nonpeptide structures are called peptidomimetics.

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80 Substantial progress has been made in the development of non-peptide molecules for the melanocortin receptors. One of the first reported nonpeptide ligands for the melanocortin receptors were based upon the beta-turn ( -turn).152 The minimal sequence required to illicit a biologi cal response is the “Phe-Arg-Trp” tripeptide, which is presumed to display a -turn conformation. A library of 951 -turn mimics based on the tripeptide was screened for agonist activity at the melanocortin receptors.152 Beta-turn mimetics containing Phe, Trp or naphthylalanine (Nap) in the i + 1 and i + 3 positions, and DLys, DArg or DPro in the i + 2 resulted in micr omolar mMC1R agonists.152 Results were consistent with a model obtain ed from homology mo lecular modeling that identified an hydrophobic and one electrostatic pocket for binding to the mMC1R (Figure 5-1).152,153 These compounds were approximately 200-fold less potent than the linear Ac-His-DPhe-Arg-Trp-NH2 tetrapeptide fragment that resulted in nana molar agonist activity at the mMC1-5R54, indicating that a fourth residue is required for potency. Based on this information, a novel thioethe r cyclized scaffold was used to mimic the -turn with four positions of diversity similar to the tetrapeptide.154 A series of nineteen compounds were screened for agonist activity at the mous e melanocortin receptors. Several compounds were identified with agonist activity, a nd three of the identified compounds were completely devoid of a basic residue capable of mimicking the Arg residue of melanocortin peptides. The mMC1R selective compound (15to 50-fold) was devoid of a basic residue. The fact that this compound is active suggests that an electrostatic interaction was not required for receptor activation. Tw o other compounds support the observation, indicating that the aromatic residues are more important for activity. Heizmann et al. screened a large combinator ial library of 328,509 trip eptoids at the MC1

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81 receptor.155 They found several novel compounds with binding affinity to the MC1R; however, all peptoids with affinity to the MC1R all shared the same structural feature of aromatic residue-basic residue-aromatic residue.155 These data call into question the importance of the Arg or basic residue in receptor activation, and suggested that the hydrophobic side chains may be more importa nt than the presence of an Arg basic residue. N H H N O N H O O HN H2N NH2NH+1 N H NH C-O O-1 O O-1 O O-1 HOGlu92 Asp 115 Asp119 Phe255 Phe193 Phe177 Tyr180 And/or Tyr181 DPhe-Arg-Trp Tripeptidyl sequence Hydrophilic binding pocket Hydrophobic/ binding pocket Phe194 His 258 Hydrophobic/ binding pocket N H H N O N H O O HN H2N NH2NH+1 N H NH C-O O-1 O O-1 O O-1 HOGlu92 Asp 115 Asp119 Phe255 Phe193 Phe177 Tyr180 And/or Tyr181 DPhe-Arg-Trp Tripeptidyl sequence Hydrophilic binding pocket Hydrophobic/ binding pocket Phe194 His 258 Hydrophobic/ binding pocket Figure 5-1. Illustration of the putative DPhe-Arg-Trp amino acids interaction with the mouse MC1R In addition to the above studies, several groups have recently reported the design and synthesis of novel non-peptide ligands for the melanocortin receptors.156-163 Most of these ligands are based upon recu rring structural features fo und in melanocortin peptides.

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82 Sebhat et al. reported one of the first highly potent and hMC4 receptor selective nonpeptide based on the 4-substituted 4-cycloh exylpiperidine template (Figure 5-2A).156 The compound had low binding affinity and agonist activity at the hMC4 and rMC4R with selectivity of 300to >3000-fold over the other receptors. However, this compound had binding affinity and agonist activity at all four human melanocortin receptors. The compound was found to significan tly inhibit food intake.156 This compound does not contain a basic moiety that can mimic the Arg side chain, although the compound is highly potent at the MC4R. The data further supports the suggesti on that the spatial arrangement of the hydrophobic groups is an im portant factor in molecular recognition and activation of the melanocor tin receptors. The significance of spatial arrangement of the hydrophobic residues may be inferred from a comparison of the low energy conformer of this small molecule with the low energy conformer of MTII. The orientation of the hydrophobic functionalities of this compound is very similar to the orientation of hydrophobic side chains in the low energy conformer of MTII.156 Compounds based on a 4-substitu ted 4-phenylpiperidine templa te similar to the above compound have been reported as highly pot ent and selective nonpe ptide MC1R agonist (Figure 5-2B).162 These compounds all lacked a basic arginine-like moiety and were all agonists at the MC1R and MC3-5R. Furt her support for the importance of the hydrophobic moiety over the basic moie ty was shown by a 2,3-diaryl-5-anilino[1,2,4]thiadiazole MC4R selective nonpe ptide (Figure 5-2C).158 This compound bound to the MC4R with nanamolar affinity when administered intraperitoneally inhibited food intake in rats.

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83 NO H N Cl O HN N N N N O NH OCH3ONH2O N NN N N N O FA B CN N S NH OCH3OCH3CH3BrDhMC4R EC50 = 2.1 nM hMC1R EC50 = 28 nM hMC4R IC50 = 52 nM hMC4R IC50 = 4.4 nM Figure 5-2. Potent and selective nonpeptid e compounds for the melanocortin receptors lacking a guanidinyl moiety In addition to the small-molecule ligands mentioned, other non-peptide compounds have been identified that interact with specif ic melanocortin receptors. Kulesza et al.161 reported trisubstituted tetrahydropyrans that bind to the MC4R with affinities similar to those of the DPhe-Arg-Trp-NH2 tripeptide. Mutulis et al.159,160 used both N-alkyl amino acid derivatives and reductive amina tion products to obtain nonpeptide compounds that bind with micromolar affinities to the MC1R and MC3R–MC5R. Like the small-molecule agonists of the me lanocortin receptors, non-peptide molecules have recently been reported that bind to the MC 4R and antagonize the activity of -MSH. Using two peptoid scaffolds, Thompson et al. designed compounds that mimic the core Arg-Phe-Phe sequence of AGRP. One of the peptoids

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84 was a functional antagonist at the MC4R.157 Arasaingham et al. pursued the design of non-peptide ligands with the ability to i nhibit AGRP binding to the MC4R based on (1-aryl-2-piperazinylethyl)piperazine scaffold (Figure 5-2D).163 The goal of the study was to inhibit AGRP interactions with the MC4R wit hout interfering with agonist activation of receptor, however, the compounds were determ ined to inhibit the activity of both AGRP and the endogenous agonist -MSH. The absence of an “argi nine-like” or basic residue in most of the reported nonpeptide ligands indicates that a basic functionality may not be necessary for ligand-recept or interaction. On the ot her hand, the “arginine-like” functionality is present in a number of these nonpeptides with melanocortin receptor activity. These data suggest th at the conformation of the lig ands during interaction with the receptor may be more important than the presence or absence of a basic functionality. SAR of Nonpeptide Ureas Based on a Linear Tripeptide The studies discussed above illustrate th e progression from pe ptide ligands to nonpeptide ligands for the melanocortin recep tors. Many of the above compounds have improved properties, such as potency, selectiv ity, and bioavailabilit y, as compared with the properties of lead pep tides. The nonpeptide compounds have provided experimental evidence to support the hypothesis regarding the bioactive conformation of peptide ligands152,154 and have linked specific melanocor tin receptors with physiological functions.156,162 The absence of basic functional ities in many of the non-peptide compounds suggest that the guanidine group found in the common ‘‘core’’ sequence of melanocortin peptides may not be essential to activity, if the hydrophobic moieties are in the correct spatial arrangemen t. The compounds have demonstrated that small molecule ligands for the melanocortin receptors are a viable option in the design of melanocortin

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85 ligands for therapeutic applica tions. The advances in development of potent and selective non-peptide ligands will enhance understanding the exact physiological roles of receptor subtypes in the melanocortin receptor family. Using the above information and the fact that the minimal fragment of the melanocortin ligands to illicit a pharmaco logical response were Ac-DPhe-Arg-Trp-NH2, a tripeptide library was designed. The six tr ipeptides were designed with Phe-Lys-Trp amino acid residues by varying their position wi thin the sequence. The Lysine in the sequence was used to replace the arginine residue, keeping the positive charge but losing the guanidinyl moiety. The screening of the six tripeptides identified the Phe-Trp-Lys-NH2 sequence as the best possible arrangement with micromolar agonist activity at the mMC1R, mMC3R, mMC5R and slight agonist activity at the mMC4R (Table 5-2). Using the side chains of Phe-Trp-Lys, a fourt een-member nonpeptide libra ry were designed and synthesized.164 A linear template was used to mimic the flexibility of the peptide and the amide bond between Phe and Trp replaced wi th a urea linkage to aid in avoiding degradation (Figure 5-3) similar to a templa te used for somatostatin receptor subtype selective analogs.165,166 The lead compound 53 (Figure 5-3) was synthesized with the side groups of Phe-Trp-Lys and characterized at the mouse melanocortin receptors. Compound 53 resulted in high micromolar activ ity at the mMC1R, mMC4R and mMC5R with no agonist activity at the mMC3R (Table 5-2). The results validated the linear urea template as a viable design fo r melanocortin receptor ligands. Compound 53 can easily be divided into three pa rts of diversity (Figure 5-3) for further structure-activity relationship studies. The aim of the study was to design a nonpeptide library patterned after the lead compound with the goal of discovering trends

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86 with increase or decrease pot ency, receptor subtype selectivity and novel pharmacology at the mouse melanocortin receptors. The study was undertaken to examine the role of aliphatic chain length, substituted aromatic mo ieties, natural and unnatural amino acids in the three position of diversity for SAR a nd selectivity at th e mouse melanocortin receptors. The monomers used for each posit ion of diversity are shown in Figure 5-4. HN H2N O NH O NH NH2O NH2HN H N O NH2H N HN OAmine Amino Acid Diamine R3R2R152 53 Figure 5-3. Illustration of the nonpeptide ur ea template based on the tripeptide and disconnection of the lead molecule into three sets of subunits used for SAR studies Results Chemical Synthesis and Characterization The compounds reported herein were synthesized using standard fluorenylmethyloxycarbonyl (Fmoc)93,94 chemistry and a parallel synthesis strategy on an automated synthesizer (Advanced Chemtech 440MOS, Louisville, KY). The nonpeptides was synthesized as described in Chapter 7 on a semi-automated synthesizer (Lab Tech, Louisville, KY) or manually. Both pep tides and nonpeptides were purified to homogeneity using semi-preparative reversed phase high pressure liquid chromatography (RP-HPLC). The purity of the analogues wa s assessed by mass spectrometry, analytical RP-HPLC in two diverse solvent systems (Table 5-1) and one-dimensional Nuclear Magnetic Resonance (1H-NMR) (Appendix A).

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87 Table 5-1. Analytical data fo r tripeptide and urea analogues 47-66 Peptide Sequence HPLC k’ (system 1) HPLC k’ (system 2) % purity m/z (M, calcd) m/z (M + 1, expt) 47 Lys-Trp-Phe-NH2 4.4 7.4 >98 478.6 479.7 48 Lys-Phe-Trp-NH2 3.9 7.0 >98 478.6 479.7 49 Trp-Phe-Lys-NH2 3.4 5.8 >99 478.6 479.7 50 Trp-Lys-Phe-NH2 4.1 7.7 >99 478.6 479.5 51 Phe-Lys-Trp-NH2 4.0 7.4 >99 478.6 479.7 52 Phe-Trp-Lys-NH2 3.0 5.4 >99 478.6 479.7 R1 R2 R3 53 Butyl L-Trp Benzyl 5.5 8.6 >98 407.2 408.8 54 Propyl L-Trp Benzyl 4.9 8.7 >99 393.5 394.7 55 Pentyl L-Trp Benzyl 5.3 9.3 >99 421.5 422.1 56 Hexyl L-Trp Benzyl 5.1 9.0 >99 435.6 436.3 57 3-methylbenzyl L-Trp Benzyl 5.3 9.0 >97 455.6 456.6 58 4-methylbenzyl L-Trp Benzyl 5.9 9.3 >99 455.6 456.7 59 4-methylpiperidinyl L-Trp Benzyl 5.4 8.4 >99 433.6 433.6 60 Butyl L-Trp Phenethyl 5.4 9.7 >99 421.5 422.2 61 Butyl L-Trp 1,2,3,4tetrahydroisoquinoline 6.2 9.8 >99 433.6 434.6 62 Butyl L-Trp 1-benzylpiperidinyl 4.2 6.7 >99 490.6 491.9 63 Butyl L-Trp 4-benzylpiperidinyl 7.5 11.4 >99 475.6 476.8 64 Butyl L-homophe Benzyl 5.6 9.4 >99 382.5 383.8 65 Butyl L-Tyr(Bzl) Benzyl 7.6 11.3 >99 474.6 475.8 66 Butyl D-Trp Benzyl 5.5 8.6 >98 407.2 408.7 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 ac id/water and a gradient to 90% methanol over 35 min). min). An analytical Vydac C18 colu mn (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. Biological Evaluation Characterization of the tripeptides, 47-52 identified peptide 52 with the ideal orientation of the three resi dues for agonist activity at th e mouse melanocortin receptors (Table 5-2). Peptide 52 was a micromolar agonist at the MC1R, MC3R, MC5R and a slight agonist at the MC4R. The side chains of peptide 52 was used to synthesize compound 53 to validate whether melanocortin recep tor activity will be maintained going from a tripeptide to a urea based small organic molecule.

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88 H2NNH2H2N H2N H2N NH2NH2NH2NH2H2N NH2H2N NH H2N1,3-Diaminopropane 1,4-Diaminobutane 1,5-Diaminopentane Hexamethylenediamine m -Xylylenediamine 4-(aminomethyl)piperidine p -XylylenediamineR1,DiamineH N HO O HN Fmoc HN HO O Fmoc O OH NH Fmoc OR2, Amino acidFmoc-Homophenylalanine Fmoc-Lor D-tryptophan Fmoc-Tyrosine(Obenzyl)NH2N H2NBenzylamine PhenethylamineNH2HN HN1-Benzyl-piperidin-4-ylamine 4-Benzyl-piperidine 1,2,3,4-Tetrahydro-isoquinolineR3, Amine Figure 5-4. Structures of m onomers used at each positi on of diversity in the urea nonpeptide library

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89Table 5-2. Functional activity of the tripeptide and urea nonpeptide analogues 47-66 at the mouse melanocortin receptors H N O N H N H O H2NR1R3R253HN EC50 (nM) Peptide Sequence mMC1R Fold diff. mMC3R Fold diff. mMC4R Fold diff mMC5R Fold diff. -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe -Arg-Trp-Gly-Lys-Pro-Val-NH2 0.3 0.04 1.2 0.3 2.5 0.3 1.5 0.5 NDP-MSH Ac-Ser-Tyr-Ser-Nle-Glu-His -DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 0.01 0.004 0.08 0.02 0.1 0.01 0.2 0.03 47 Lys-Trp-Phe-NH2 2840 170 > 100000 > 100000 Slight agonist 48 Lys-Phe-Trp-NH2 Slight agonist > 100000 > 100000 Slight agonist 49 Trp-Phe-Lys-NH2 3125 715 Slight agonist Slight agonist Slight agonist 50 Trp-Lys-Phe-NH2 Slight agonist > 100000 Sli ght agonist Slight agonist 51 Phe-Lys-Trp-NH2 Slight agonist > 100000 > 100000 > 100000 52 Phe-Trp-Lys-NH2 3437 2053 6545 314 Slight agonist 4889 2653 R1 R2 R3 53 Butyl L-Trp Benzyl 55000 227001 >100000 23700 6400 1 30500 1 54 Propyl L-Trp Benzyl 51200 190001 >100000 >100000 39900 8800 1 55 Pentyl L-Trp Benzyl 14800 7300 -4 >100000 >100000 20000 3100 -1.5 56 Hexyl L-Trp Benzyl 22300 8800 -2.5 >100000 >100000 >100000 57 3-methylbenzyl L-Trp Benzyl 28000 7700 -2 >100000 >100000 >100000 58 4-methylbenzyl L-Trp Benzyl 400 200 -138 10700 1000 400 200 -59 4900 4800 -6 59 4-methylpiperidinyl L-Trp Benzyl 5200 2700 -11 4400 2800 >100000 8200 7900 -4 60 Butyl L-Trp Phenethyl >100000 >100000 >100000 >100000 61 Butyl L-Trp 1,2,3,4-tetrahydroisoquinoline 7900 1000 -7 1700 1000 3400 1900 -7 4900 4800 -6 62 Butyl L-Trp 1-benzylpiperidinyl 5000 2000 -11 4400 2800 2800 2500 -8 8200 7900 -4 63 Butyl L-Trp 4-benzylpiperidinyl 20000 4800 -3 >100000 >100000 28900 6800 1 64 Butyl L-homophe Benzyl >100000 >100000 >100000 >100000 65 Butyl L-Tyr(Bzl) Benzyl 20500 10100-3 >100000 >100000 14400 3600 -2.1 66 Butyl D-Trp Benzyl 42400 158001 >100000 23700 7400 1 32800 19100 1 Indicated errors represent the st andard error of the mean determ ined from at least three independ ent experiments. Slight agonis t denotes that some stimulatory response was observed but not enough to determine an EC50 value. >100000 indicates no agonist activity at up to 100000 nM concentrations

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90 Compound 53 possessed high M mMC1R, mMC4 R and mMC5R agonist activity and no agonist activity at up to 100000 nM at the mMC3R. A library of fourteen compounds was subsequently prepared making changes at the R1, R2 and R3 positions (Figure 5-3) with the inten tion of creating more potent a nd selective compounds for the melanocortin receptor subtypes. Table 5-2 su mmaries the tripeptide and urea nonpeptide agonist pharmacology at the mouse melanocortin MC1and MC3-5 receptors . Mouse melanocortin-1 receptor Compounds 54-59 with modifications at the R1 (diamine) position, in relation to the lead compound, 53 were all agonist at the MC1R. Shortening or lengthening the methylene chain ( 54-56 ) at the R1 resulted in analogues that were equipotent compared to 53 . However, compared to the control peptide, 52 these analogues were 4to 15-fold less potent. Compounds 57-59 contain cyclo-hexyl ri ngs that were used to determine whether conformational constraints will have any effect at this position. Compound 57 with a benzyl aromatic ring and a methyl group at the 3 position resulted in high micromolar activity at the MC1R equipotent to 53 (within experimental error). Interestingly when the methyl group was placed at the 4 position of the benzyl ring it resulted in nanamolar potent analogue ( 58 ) with 138-fold improve potency compared to 53 . When the benzyl ring in 58 was modified to a piperidine ring, compound 59 , there was a 13-fold decreased in potency compared to 58 . Compounds 60-63 had modifications made at the R3 (amine) group, while R1 and R2 were retained, as in compound 53 (Figure 5-3). When the benzyl group of 53 was changed to phenethyl, 60 , a complete loss of activity at up to 100 M concentrations was seen at the MC1R. When R3 was 1,2,3,4-tetrahydroisoquinoline (compound 61 ), 6fold increased potency re sulted at the MC1R compared to 53 . Compound 62 with a benzyl group attached to a pipe ridine ring at the 1-position, resulted

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91 in increased (11-fold) potency compared to 53 . Compound 63 with the benzyl group attach to the piperidine ri ng at the 4-position, however, wa s 4-fold less potent than compound 62 . Compounds 64-66 had modifications at the R2 (amino acid) group, while retaining the R1 and R3 groups of compound 53 (Figure 5-3). None of the three compounds showed improved agoni st activity compared to 53 . Compound 64 containing the R group of L-homophenylalanin e lost activity at the MC1R. Mouse melanocortin-3 receptor All aliphatic R1 analogues containing thr ee to six methylene CH2 between the two amino groups (compounds 53-56 ) had no activity up to 100 M at the MC3R. Compounds 57-59 contain cyclo-hexyl rings that were used to determine whether conformational constraints will have any effect at this position. Compound 57 with a benzyl aromatic ring and a methyl group at the 3 position had no agonist activity. Interestingly when the methyl group was placed at the 4 position of the benzyl ring it resulted in micromolar agonist ( 58 ). Compound 59 with a piperidine ring replacing the benzyl ring, resulted in agoni st activity equipotent to 58 (within experimental error). Compounds 64-66 had modifications at the R2 (amino acid) group, while retaining the R1 and R3 groups of compound 53 (Figure 5-3). None of the three compounds had agonist activity at the MC3R. Mouse melanocortin-4 receptor Compounds 54-59 with modifications at the R1 (diamine) position, in relation to the lead compound, 53 all lost agonist at the MC4R except compound 58 . Compound 58 (EC50 = 400 nM) is 59-fold more poten t at the MC4R compared to 53 . Compounds 60-63 had modifications made at the R3 (amine) group, while R1 and R2 were retained, as in compound 53 (Figure 5-3). When the benzyl group of 53 was changed to phenethyl, 60 , a

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92 complete loss of activity at up to 100 M c oncentrations was seen at the MC4R. When R3 was 1,2,3,4-tetrahydroisoquinoline, 61 , or a benzyl group attached to a piperidine ring at the 1-position, 62 , there was a 7fold increased in potency at the MC4R compared to 53 . Compound 63 with the benzyl group attach to the piperidine ring at the 4-position, however, had no agoni st activity. Compounds 64-66 had modifications at the R2 (amino acid) group, while retaining the R1 and R3 groups of compound 53 (Figure 5-3). Compounds 64 and 65 both had to agonist activity and compound 66 was equipotent compared to 53 at the MC4R. Mouse melanocortin-5 receptor Compounds 54-59 were modified at the R1 (diamine) position, in relation to the lead compound, 53 . Shortening or lengthening th e methylene chain by one CH2 group ( 54-55 ) at the R1 resulted in analogues that were equipotent compared to 53 . However, further lengthening of the methylene chain ( 56 ) resulted in lost of potency at the MC5R. Compounds 57-59 contain cyclo-hexyl rings that were used to determine whether conformational constraints will have any effect at this position. Compound 57 with a benzyl aromatic ring and a methyl group at the 3 position had no agonist activity. Interestingly, when the methyl group was place d at the 4 position of the benzyl ring it resulted in micromolar agonist activity ( 58 ) with 6-fold increased potency compared to 53 . Compound 59 with a piperidine ring replacing the benzyl ring, resulted in agonist activity equipotent to 58 (within experimental error). Compounds 60-63 had modifications made at the R3 (amine) group, while R1 and R2 were retained, as in compound 53 (Figure 5-3). When the benzyl group of 53 was changed to phenethyl, 60 , a complete loss of activity at up to 100 M c oncentrations was seen at the MC5R. When R3 was 1,2,3,4-tetrahydroisoquinoline (compound 61 ), 6fold increased potency resulted at

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93 the MC4R compared to 53 . Compound 62 with a benzyl group attached to a piperidine ring at the 1-position, resulted in slightly increa sed (4-fold) potency compared to 53 . Compound 63 with the benzyl group attach to the piperidine ring at the 4-position, however, was 4-fold less potent than compound 62 . Compounds 64-66 had modifications at the R2 (amino acid) group, while retaining the R1 and R3 groups of compound 53 (Figure 5-3). None of the three compounds showed improved agonist activity compared to 53 . Compound 64 containing the R group of Lhomophenylalanine had no agonist activity at the MC5R. Discussion This study was undertaken to prepare sm all organic molecules that possessed agonist activity at the melanoc ortin receptors. The MC3 and MC4 receptors are expressed in the brain22,29 and are implicated in regulati ng weight and energy homeostasis.40,74,167 Comparison of the R1 aliphatic diamines identified four methylene groups as the optimal spacing between the amine groups to possess MC4R agonist activity. Compounds 53-56 shows that as the aliphatic chain length of the diamine (R1) were lengthened or shortened from four methylene groups, all agonist activ ities at the MC4 recep tor was abolished and none of these analogues were MC3R agonist . However, the length of the aliphatic methylene chain had no effect on the MC1R because all the analogues ( 53-56 ) were equipotent at this receptor. When a ring system was used at the R1 position, MC4R agonist activity resulted when the aromatic ring had a m odification at the 4-position (compounds 57, 58 and 59) . However compound 59 with a piperidine ring replacing the benzyl group of compound 58 lost agonist activities at th e MC4R but retained reduced agonist potency and selectivity at the MC1, MC3 and MC5 receptors. Based on the results the benzyl ring with the methyl group at the 4-position ( 58 ) was preferred at the

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94 MC1, MC4 and MC5 receptors. The MC3R prefe rred the piperidine ring with the methyl at the 4-position ( 59 ) over analogue 58 . Compounds 64 and 65 lost agonist activity at the MC3 and MC4 receptors when the R2 subunit was modified. Replacing Trp w ith homophe resulted in an analogue ( 64 ) that had to agonist activity at four re ceptor subtypes tested. The D-tryptophan R2 subunit, (compound 66 ) retained agonist activity at th e MC1R, MC4R and MC5R but was equipotent (within expe rimental error of 53 ) at these receptor. Like the lead, 53 , compound 66 had no activity at the MC 3R. These data supported the hypothesis that an indole ring is important for maintaining agonist activity at the MC3R and MC4R in this small molecule template because replacing the indole ring of tryptophan resulted in complete lost of agonist activit ies at these two receptors. Changes made at the R3 (amine) subunit produced varied results. All four melanocortin receptors tested lost ag onist activities when the methylene CH2 link was extended to two methylene groups, compound 60 . Removal of the methylene group completely, also resulted in lost of agonist activities at either all four melanocortin receptors or two of the four tested (compounds 61-63 ). However, MC3R and MC4R agonist activities were dependent upon whether the benzyl ring was attached directly to the nitrogen ( 62 ) or at the 4-position of the piperidine ring ( 63 ). Attachment directly to the nitrogen in the piperidine ring (compound 62 ) produce high M EC50 values at the MC3 and MC4 receptors but no activity was obser ved at these receptors when the benzyl group was attached at the 4-position (compound 63 ). Although 63 retained agonist activity at the MC1R and MC5R, 62 was 4-fold more potent at these receptors compared to 63 , respectively.

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95 Compound 58 (Figure 5-5) was the most poten t analogue at the MC1, MC4 and MC5 receptors with 27and 12-fold more selective for the MC4R and MC1R (EC50 = 400 nM) versus MC3R, and MC5R, respectivel y. Unfortunately, this analogue was not selective for one receptor subtype but it is an excellent starting point for future studies. H N O N H N H O H2N58NH mMC1R mMC3R mMC4R mMC5R 0 2000 4000 6000 8000 10000 12000400 nM 400 nME C5 0( n M )10700 nM 4900 nM 27-fold 12-fold H N O N H N H O H2N58NH mMC1R mMC3R mMC4R mMC5R 0 2000 4000 6000 8000 10000 12000400 nM 400 nME C5 0( n M )10700 nM 4900 nM 27-fold 12-fold mMC1R mMC3R mMC4R mMC5R 0 2000 4000 6000 8000 10000 12000400 nM 400 nME C5 0( n M )10700 nM 4900 nM 27-fold 12-fold Figure 5-5. Structure of equipotent potent analogue at the MC1 and MC4 receptors Summary A small focused library of urea peptidom imetics based on a tripeptide, displaying diversity at up to three positions resulted in compounds with micromolar to nanamolar agonist potencies at the mMC1R and mMC3 -5R. A nanamolar potent MC4R agonist, compound 58 , with improved potency ove r the lead tripeptide, 52 (Figure 5-6) was identified. However, this compound was not se lective for the MC4R since it had the same potency (400 nM) at the MC1R. Compound 59 was identified as being slightly selective for the MC3R (4.4 M) over the MC4R with th is compound having no agonist activity up to 100 M at the MC4R. These results may be useful in the further design of potent and selective non-peptide ligands for the melanocortin receptors.

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96 mMC4R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0.00 0.25 0.50 0.75 1.00 1.25NDP-MSH 58 52 Log concentration compound [M]-galactosidase normalized to protein and forskolin Figure 5-6. Illustration of the tripeptide 52 , Phe-Trp-Lys-NH2 possessing slight agonist activity and the nonpeptide, 58 possessing full nanamolar agonist activity at the mMC4R. The peptide NDP-MSH is included as a control to illustrate the maximal response observed for full agonist.

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97 CHAPTER 6 STRUCTURE-ACTIVITY RELATIONS HIPS OF THE MELANOCORTIN TETRAPEPTIDE Ac-HIS-DPHE-ARG-TRP-NH2 AT THE MOUSE MELANOCORTIN RECEPTORS: MODIFI CATION OF THE ARG SIDE CHAIN All peptides were designed, synthesized, pur ified, and analytically characterized by Christine G. Joseph under the supervisi on of Dr. Carrie Haskell-Luevano. The pharmacology studies were carried out by Nick Sorensen and Michael Wood, both member’s of the Haskell-Luevano laboratory group. Data analysis was performed by Dr. Carrie Haskell-Luevano. NMR data was obtai ned by Christine Joseph at the Advanced Magnetic Resonance Imaging and Spectroscopy (A MRIS) facility in the McKnight Brain Institute of the University of Flor ida with guidance from Mr. Jim Rocca. Introduction Since the beginning of the 1990s after the cloning of the melanocortin receptors, they have become the center of a large amount of research by both academic and industrial laboratories. The me lanocortin receptor family participate in a vast array of physiological functions, such as, skin pigm entation, steriodogenesis , weight, and energy homeostasis.167-170 The MC3R and MC4R involvement in weight and energy homeostasis and in particular the MC4R involvement in food intake are the main targets in research laboratories. Both endogenous a nd synthetic melanocortin ligands have been lead compounds in many structure-activity (SAR) studies to improve potency and selectivity. The studies discussed below ex emplify the rational design processes of peptide research and reveal the insight thes e studies have provided to the melanocortin field. Amino acid numbering throughout this ch apter refers to the corresponding position

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98 of the amino acid residue in the sequence of -MSH (Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2). Truncation Studies Truncation is one of the first steps taken once a peptide lead has been established, because it aids in the identif ication of amino acid residues that contribute to molecular recognition and receptor stimulation. Structure-activity relationships for –MSH and the highly-potent analogue NDP-MSH (Ac-Ser1-Tyr2-Ser3Nle4-Glu5-His6DPhe7-Arg8-Trp9Gly10-Lys11-Pro12-Val13-NH2) have been investigated usi ng amphibian and reptilian skin bioassays and mammalian melanoma cell cultures to determine the minimal sequence required to illicit a pharmacological response.54-56,171-173 These studies involved the binding affinity and biological activity of the fragments with successive deletion of amino acids from each terminus (Nand C-te rminal). Peptide fragments activity in the classical frog ( Rana pipiens ) and lizard ( Anolis carolinensis ) skin bioassays, was monitored by quantifying the response through the amount of skin darkening that occurs. The minimal sequence required for biological activity was determined to be Ac-His6Phe7-Arg8-Trp9-NH2 for –MSH55,56,173 and Ac-DPhe7-Arg8-Trp9-NH2 for NDP-MSH using these bioassays.55,172 Using the cloned mouse a nd human melanocortin receptors (MC1R, MC3R-MC5R), truncation of NDP-MSH was performed to determine if results from the cloned receptors correlate with th e previous results using the classical skin bioassays.54,174 Results at both the mouse and human receptors identify Ac-DPhe-ArgTrp-NH2 as the minimal NDP-MSH sequence re quired for activity with >100-fold reduced potency, but addition of histidine si gnificantly increase potency at each of the four receptors. In the frog skin bioassay, the truncated –MSH fragments revealed that

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99 residues 4,10, and 12 are required for potency and that residues 1-3, 5, 11, and 13 has very little to no effect on potency.173 These results were also supported in the lizard skin biossay.56 These studies identi fied residues 4-12 of –MSH and residues 4-9 of NDP-MSH as the minimal fragments required to re tain potency equivalent to their respective lead peptides. Truncation of –MSH and NDP-MSH resulted in the identification of a core sequence (Ac-Phe-Arg-Trp-NH2 for -MSH and Ac-DPhe-Arg-Trp-NH2 for NDP-MSH) that is required to elicit measurable biological activity; th ey contain important amino acids that are required to retain equipot ency to the parent peptide; and the peptides contain amino acids that contribute mini mally to the potency of the ligands. Alanine Scans The next step is to identify amino acid si de chains participating in ligand-receptor interaction while leav ing the stereochemistry of th e peptide backbone unchanged. The importance of each residue is determined by an L-alanine scan where individual amino acid in the lead peptide ligand is systematica lly replace with an alan ine. Identification of important side chain residues would be usef ul when designing ligands with specific activity at a particular receptor system. The influence of single amino acid replacements by alanine on the binding affin ity and biological activity of -MSH utilizing B16 murine melanoma cells (putative MC1R),171 and more recently -MSH (Tyr1-Val2-Met3-Gly4His5-Phe6-Arg7-Trp8-Asp9-Arg10-Phe11-Gly12-OH) using the cloned human MC3R-MC5R131has been studied systematically. Alanine scan of -MSH in the B16 mouse melanoma cells revealed that the three te rminal amino acids at both the Cand N-terminal were not necessary for binding or activity.175 The core sequence of residues 4 to 9 was required for receptor binding and triggeri ng a biological response. Each residue

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100 within the His-Phe-Arg-Trp core sequence was shown to be essential with Phe, Arg, and Trp having a large reduction in potency when replaced with alanine compared to the His residue. Similar results were obtained with -MSH at the human receptors (MC3-5R). The last four residues at the C-terminal we re not important determinants of biological activity and selectivity.131 Both binding affinity and biological activity decreased significantly when His5, Phe6, Arg7, and Trp8 were replaced with alanine. The alanine scan of both -MSH and -MSH highlights the importan ce of the His-Phe-Arg-Trp sequence in both endogenous melanocortin lig ands in receptor binding and agonist activity.131,171 Melanocortin His-DPhe-Arg-Trp Tetrapeptide Sequence The SAR studies just discussed support the hypothe sis that the His-DPhe-Arg-Trp sequence represents the key structural features required for functional activity at the melanocortin receptors.54,174 In a recent study in our laboratory, substitution at the Arg position with neutral, basic or acidic amino aci d side chains showed that removing the arginine guanidinyl side chai n resulted in decreased mela nocortin receptor potency, and that the side chain was not necessary fo r melanocortin receptor agonist activity.176 It was hypothesized that pharmacological characteri zation of a set of carefully designed analogues based on this sequence could provi de useful information for future development of melanocortin ligands with improved properties. The study presented next was undertaken to examine the role of various urea, thiourea, and carbamate functional modification at the arginine pos ition of the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 for structure-activity-relationships a nd selectivity properties at the mouse melanocortin receptors. The aim of the study wa s to identify trends that result in an

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101 increase or decrease in potency, receptor subtype selectivity, and novel pharmacology at the melanocortin receptors. Previous Modifications of the Arg8 Position in Melanocortin Receptor Ligands The Arg side chain contained within th e conserved melanocortin agonist ligand “His-Phe-Arg-Trp” sequence has been previous ly thought to play an important role in melanocortin receptor stimulation. Replacement of Arg8 by Ala8 in the native -MSH peptide agonist resulted in 2000-fold decrea sed binding affinity in mouse B16 melanoma cells (MC1R).171 In -MSH, replacement of Arg7 with Ala resulted in > 50-fold decrease agonist activity at the human MC3-5 receptors,131 while replacement with DArg resulted in 130-fold decreased agonist ac tivity at the hMC3R and total lo st of activity at the hMC4 and hMC5 receptors.132 Substitution of Arg8 by Pro8 in the MTII (Ac-Nle4-c[Asp5-His6-DPhe7-Arg8-Trp9-Lys10]-NH2) peptide template (a cyclic analog of -MSH) resulted in only 2 to 7% cAMP stimulation up to 2 M at the hMC3R and hMC4R, respectively; and 330to 10,000-fold reduced binding affinity at hMC3-5R.177Substitution of the Arg8 by Ala in the MTII template resulted in 1620-fold hMC3R, 1370-fold hMC3R reduced binding affinity, and 138and 262-fold decreased potency at the human MC3 and MC4 receptors.178 Substitution of Glu8 for Arg8 in MTII resulted in only 10to 100-fold less potency than the parent compound in the cAMP accumulation assays at hMC3-5R;179 whereas substituting Glu8 for Arg8 in the -MSH peptide resulted in 4000and 2500-fold decreased agonist potency at the hMC3R and hMC4R.179 Although potency was reduced when Arg was replaced with Glu in MT II, the study unequivocally shows that the side chain of Arg8 was not essential for efficient interactions of MTII with the melanocortin receptors (particularl y the MC4R). On the other hand, Arg8 was essential for

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102 -MSH interaction with the melanocor tin receptors. The idea that Arg8 was not essential for MTII interaction with the melanocortin receptors was supported further when it was replaced with Lys and there was only a 123and 10-fold decreased potency at the hMC3 and hMC4 receptors, respectively.179 The above results indicated that Arginine appears to play a significantly smaller role in li gand-receptor interac tion between MTII and melanocortin receptors but play s a much bigger role between -MSH and melanocortin receptors. However, other studies of melanocortin ligand substitutions at the Arg position suggest that this side chain may not be as cr itical for melanocortin receptor activation as previously thought.179-181 For example, Glu8 substitution for Arg8 in the NDP-MSH peptide template only resulted in 90-fo ld decreased hMC3R and hMC4R potency, compared to NDP-MSH at these receptors.179 This suggests that, in combination with the DPhe7, the Arg8 side chain plays a less dramatic role for melanocortin receptor potency.179 Also the replacement of Arg8 with Lys8 in NDP-MSH resulted in 10to 37-fold decreased binding affinity at the hMC1R and hMC3-5R,181 further supporting that the Arg8 is less important for interaction with the melanocortin receptors. A study carried out on a linear pentapeptide (Bu-His-DPhe-Arg8-Trp-Gly-NH2) in which Arg8 was replaced with arginine surroga tes, cyanoguanidine, or acylguani dine, resulted in equipotent agonists at the hMC1R and hMC4R.180 The lack of a positive charge on these two analogues indicates that the positive charge of Arg8 is not essential for ligand-receptor interaction and could be repl aced without loss of functi on at hMC1R and hMC4R. This study was undertaken to examine th e effect that modifications at Arg8 of Ac-His-Phe-Arg-Trp-NH2 would have on mouse MC3-5 receptors using a novel template.

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103 Results and Discussion Chemical Synthesis and Characterization The peptides reported herein were synthesized using standard fluorenylmethyloxycarbonyl (Fmoc)93,94 chemistry and a parallel synthesis strategy both manually and on a semiautomatic synthesizer (Advanced ChemTech Labtech, Louisville, KY). The side chain amino group of the Lys or Orn residue was allowed to react with an aryl or alkyl isocyanate or is othiocyanate to produce the fina l urea or thioureacontaining tetrapeptide. The peptides were purified to homogeneity using semi-preparative reversedphase high pressure liquid chromatography (RPHPLC). The purity of these peptides was assessed by mass spectrometry (Table 6-1), an alytical RP-HPLC in two diverse solvent systems (Table 6-1), and one-dimensional 1H NMR (Appendix B). Biological Evaluation Table 6-2 summarizes the pharmacology at the mouse melanocortin receptors, mMC3R, mMC4R, and mMC5R of the tetrapep tides modified at the Arg position of the tetrapeptide template, Ac-His-DPhe-Arg8-Trp-NH2, prepared in this study. The Arg residue of Ac-His-DPhe-Arg-Trp-NH2 was substituted with Lys, Orn, homocit, Lys(Z) and Lys(Aloc). The Urea a nd thiourea analogues we re synthesized after removal of aloc from Orn or Lys that were th en reacte d with isocyanates or isothiocyanates (Figure 6-1). The development of this novel series of melanocortin agonists is based on the suggestion that the position charge or the ba sic gaunidinyl functionality is not necessary in nonpeptide ligands to be melanocortin agonists.152,154,156,158-160,163,182 Nonpeptide ligands lacking the arginine functionality has been reported as potent and selective for the MC1R162 and MC4R.156 In this chapter,we report the results of structure-activity

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104 relationship of these melanocortin-based te trapeptides bearing a urea, thiourea or carbamate in the critical side chain on the mMC3-5 receptors. Table 6-1. Analytical data for the Arg modified tetrapeptides 67-97 Peptide Sequence HPLC k’ (system 1) HPLC k’ (system 2) % purity m/z (M, calcd) m/z (M + 1, expt) 67 Ac-His-DPhe-Arg-Trp-NH2 3.9 6.5 >98 685.8 686.3 68 Ac-His-DPhe-Lys-Trp-NH2 3.9 6.7 >99 657.8 658.6 69 Ac-His-DPhe-Lys(Z)-Trp-NH2 7.0 11.1 >95 791.9 792.2 70 Ac-His-DPhe-Lys(Aloc)-Trp-NH2 6.5 10.4 >99 741.8 742.4 71 Ac-His-DPhe-homocit-Trp-NH2 4.1 7.3 >99 700.8 701.2 72 Ac-His-DPhe-Orn-Trp-NH2 4.1 7.1 >99 643.7 644.6 73 Ac-His-DPhe-Orn(benzyl urea)-Trp-NH2 6.5 10.8 >99 776.9 777.6 74 Ac-His-DPhe-Lys(benzyl thiourea)-Trp-NH2 7.3 11.7 >97 807.0 807.2 75 Ac-His-DPhe-Lys(benzyl urea)-Trp-NH2 6.7 11.2 >98 790.9 791.6 76 Ac-His-DPhe-Lys(phenyl urea)-Trp-NH2 6.8 11.2 >98 776.9 777.7 77 Ac-His-DPhe-Lys(phenyl thiourea)-Trp-NH2 7.0 11.0 >97 793.0 793.7 78 Ac-His-DPhe-Lys(cyclohexyl urea)-Trp-NH2 6.8 11.7 >99 782.9 783.7 79 Ac-His-DPhe-Lys(cyclohexyl thiourea)-Trp-NH2 7.6 12.1 >97 799.0 799.3 80 Ac-His-DPhe-Lys(ethyl urea)-Trp-NH2 5.0 9.1 >99 728.8 729.6 81 Ac-His-DPhe-Lys(ethyl thiourea)-Trp-NH2 5.7 9.7 >99 744.9 745.7 82 Ac-His-DPhe-Lys(methyl thiourea)-Trp-NH2 5.3 8.9 >98 730.3 731.6 83 Ac-His-DPhe-Lys(2-chlorophenyl urea)-Trp-NH2 7.4 12.6 >99 811.3 811.3 84 Ac-His-DPhe-Lys(4-chlorophenyl urea)-Trp-NH2 7.7 12.9 >99 811.3 811.3 85 Ac-His-DPhe-Lys(4-methylphenyl urea)-Trp-NH2 7.2 11.6 >98 790.9 791.6 86 Ac-His-DPhe-Lys(4-methoxyphenyl urea)-Trp-NH2 6.6 10.7 >99 806.9 807.6 87 Ac-His-DPhe-Lys(1-naphthyl urea)-Trp-NH2 7.5 12.0 >96 826.9 827.7 88 Ac-His-DPhe-Lys(2-naphthyl urea)-Trp-NH2 7.9 13.3 >97 826.9 827.6 89 Ac-His-DPhe-Lys(2-biphenyl urea)-Trp-NH2 8.2 12.9 >99 853.0 853.6 90 Ac-His-DPhe-Lys(4-ethoxyphenyl urea)-Trp-NH2 7.0 11.9 >98 820.9 821.2 91 Ac-His-DPhe-Lys(4-nitrophenyl urea)-Trp-NH2 7.5 11.9 >99 821.9 844.8 Na salt 92 Ac-His-DPhe-Lys(4-isopropylphenyl urea)-Trp-NH2 7.7 12.1 >97 818.4 819.2 93 Ac-His-DPhe-Lys(3-acetyl urea)-Trp-NH2 6.8 10.1 >99 818.9 818.3 94 Ac-His-DPhe-Lys(2,4-dichlorophenyl urea)-Trp-NH2 8.4 12.6 >98 845.8 845.3 95 Ac-His-DPhe-Lys(trans-2-phenylcyclopropyl urea)Trp-NH2 7.0 11.7 >95 816.9 817.4 96 Ac-His-DPhe-Lys(R-(+)-a-methylphenyl urea)-Trp-NH2 7.0 11.3 >97 804.9 805.4 97 Ac-His-DPhe-Lys(S-(-)-a-methylphenyl urea)-Trp-NH2 7.0 11.4 >99 804.9 805.4 HPLC k’ = [(peptide retention time – solvent rete ntion 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 9 % methanol over 35 min). An analytical Vydac C18 column (Vydac 218TP104) was used with a flow rate of 1.5 m L/min. The peptide purity was determined by HPLC at a wavelength of 214 nm

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105 H2N OH O NH2H2N OH O NH2H2N OH O H NO OLysNCS NCS NCS NCO Cl N C O NCO H3C NCO NCO NCO N C O NCO NCO NCO NCO O NCO NCO O2N NCO Cl Cl NCO C2H5O NCO Cl NCO Cl NCO H3CO NCO NCS NCS NCO H2N OH O H NO O H2N OH O H NNH2OOrn Lys(Aloc) Lys(Z) homocit THIOUREA ANALOGUESBenzyl isothiocyanate Phenyl isothiocyanate Cyclohexyl isothiocyanate Ethyl isothiocyanate Methyl isothiocyanate Benzyl isocyanate Phenyl isocyanate Cyclohexyl isocyanate Ethyl isocyanate 2-chloroethyl isocyanateUREA ANALOGUES2-Chlorophenyl isocyanate 4-Chlorophenyl isocyanate 4-Methylphenyl isocyanate 4-Methoxyphenyl isocyanate 2,4-Dichlorophenyl isocyanate 4-Nitrophenyl isocyanate 4-Ethoxyphenyl isocyanate 4-isopropylphenyl isocyanateR-methylphenyl isocyanate 1-Naphthyl isocyanate 2-Naphthyl isocyanate 2-Biphenyl isocyanate trans-2-phenylcyclopropyl isocyanate 3-acetylphenyl isocyanate S-methylphenyl isocyanate Figure 6-1. Structures of the am ino acids used to replace Arg in the tetrapeptide template and monomers used for formati on of urea or thiourea analogues The effect of receptor agonist activity as a function of the carbon chain length connecting the critical urea moiety to the peptide backbone was investigated by substituting Arginine in tetrapeptide 67 with lysine (Lys) and ornithine (Orn), which lengthened and maintained the side chain, re spectively. Both replacement resulted in a decrease in activity as reported previously.176 However, the Orn replacement, tetrapeptide 72 was less potent that the Lys, tetrapeptide 68 at the mMC3-5 recept ors (Table 6-2). The pharmacological data indicated significant loss of agonist activity for both compounds 68 and 72 (Table 6-2), with the ornithine derivative, 72 possessing weaker activity at the

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106Table 6-2. Functional activity of the Arg modified tetrapeptides 67-97 at the mouse melanocortin receptors EC50 (nM) Peptide Sequence mMC1R Fold diff. mMC3R Fold diff. mMC4R Fold diff mMC5R Fold diff. -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-P he-Arg-Trp-Gly-Lys-Pro-ValNH2 0.7 0.2 2.1 0.5 2.6 0.3 2.0 0.2 MT-II Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 0.02 0.002 0.1 0.02 0.04 0.007 0.3 0.03 67 Ac-His-DPhe-Arg-Trp-NH2 97 90 1 376 111 1 10 3 1 5 2 1 68 Ac-His-DPhe-Lys-Trp-NH2 229 70 2 12257 2571 33 326 23 33 380 168 76 69 Ac-His-DPhe-Lys(Z)-Trp-NH2 1042 341 11 > 100000 5800 1960 580 3743 1472 749 70 Ac-His-DPhe-Lys(Aloc)-Trp-NH2 656 247 7 > 100000 8327 2664 833 2886 1166 577 71 Ac-His-DPhe-homocit-Trp-NH2 902 516 9 23000 6800 61 4273 873 427 2024 694 405 72 Ac-His-DPhe-Orn-Trp-NH2 1885 245 19 43550 18950 116 1856 638 186 454 57 91 73 Ac-His-DPhe-Orn(benzyl urea)-Trp-NH2 8225 6475 85 14500 1600 39 1797 702 180 406 116 81 74 Ac-His-DPhe-Lys(benzyl thiourea)-Trp-NH2 221 120 2 28200 75 7587 1655 759 3210 970 642 75 Ac-His-DPhe-Lys(benzyl urea)-Trp-NH2 NA 19000 51 6210 1160 621 1710 110 342 76 Ac-His-DPhe-Lys(phenyl urea)-Trp-NH2 605 446 6 11300 30 5747 1076 575 2425 195 485 77 Ac-His-DPhe-Lys(phenyl thiourea)-Trp-NH2 36 19 -3 25300 67 5767 618 577 740 139 148 78 Ac-His-DPhe-Lys(cyclohexyl urea)-Trp-NH2 1217 384 13 > 100000 6957 1753 696 1800 360 79 Ac-His-DPhe-Lys(cyclohexyl thiourea)-Trp-NH2 244 46 3 37250 4350 99 5183 880 518 3150 351 630 80 Ac-His-DPhe-Lys(ethyl urea)-Trp-NH2 1770 190 18 71400 190 6127 1189 613 3097 818 619 81 Ac-His-DPhe-Lys(ethyl thiourea)-Trp-NH2 3125 1115 32 24450 5950 65 4233 843 423 1604 655 321 82 Ac-His-DPhe-Lys(methyl thiourea)-Trp-NH2 875 240 9 13810 5381 37 3653 700 365 413 68 83 83 Ac-His-DPhe-Lys(2-chlorophenyl urea)-Trp-NH2 872 614 9 > 100000 7703 797 770 4053 1530 811 84 Ac-His-DPhe-Lys(4-chlorophenyl urea)-Trp-NH2 524 213 5 > 100000 4740 1831 474 532 81 106 85 Ac-His-DPhe-Lys(4-me thylphenyl urea)-Trp-NH2 258 92 3 > 100000 3690 558 369 517 141 103 86 Ac-His-DPhe-Lys(4-methoxyphenyl urea)-Trp-NH2 162 15 2 45000 120 3287 377 329 1250 201 250 87 Ac-His-DPhe-Lys(1-naphthyl urea)-Trp-NH2 682 209 7 > 100000 10243 874 1024 2045 747 409 88 Ac-His-DPhe-Lys(2-naphthyl urea)-Trp-NH2 242 53 3 > 100000 2240 880 224 3184 1993 637 89 Ac-His-DPhe-Lys(2-biphenyl urea)-Trp-NH2 376 111 4 > 100000 4590 815 459 1960 403 392 90 Ac-His-DPhe-Lys(4-ethoxyphenyl urea)-Trp-NH2 NA 41633 20206 111 4958 1734 496 3068 1205 614 91 Ac-His-DPhe-Lys(4-nitrophenyl urea)-Trp-NH2 NA 20358 6482 54 4328 1787 433 1056 276 211 92 Ac-His-DPhe-Lys(4-is opropylphenyl urea)-Trp-NH2 NA 32400 86 5465 2160 547 3336 1832 667 93 Ac-His-DPhe-Lys(3-acetyl urea)-Trp-NH2 NA 41257 26106 110 5851 1869 585 1137 289 227 94 Ac-His-DPhe-Lys(2,4-dichlorophenyl urea)-Trp-NH2 NA > 100000 5550 1978 555 4346 2310 869 95 Ac-His-DPhe-Lys(trans-2phenylcyclopropyl urea)-Trp-NH2 312 3 27870 15769 74 9050 3033 905 3328 1186 666 96 Ac-His-DPhe-Lys(R-(+)-methylphenyl urea)-Trp-NH2 768 8 42600 8757 113 9793 2780 979 5621 2925 1124 97 Ac-His-DPhe-Lys(S-(-)-methylphenyl urea)-Trp-NH2 309 3 38733 16519 103 12278 3739 1228 3314 1304 663 The indicated errors represent the standard error of the mean determined from at leas t three independent experiments. NA means that the analogues were not tested at that receptor. >100000 indicate that no agonist activity was observed at up to 100000 nM concentrations

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107 mMC3-5 receptors than the lysine compound, 68 . Both compounds maintained roughly 23to 96-fold selectivity for the mMC4R and mMC5R over the mMC3 receptor compared to peptide 67 . Despite their loss in melanocortin receptor potencies, both compounds maintained their ability to s timulate agonist activities (Table 6-2). Two related tetrapeptide derivative 69 and 70, in which the Arginine was replaced with an NZ-lysine and N-aloc-lysine residue was synthesized and characterized. These derivatives possessed similar agonist activities for the mMC3-5 receptors but had decreased potency at the mMC4R and mMC5R (575to 850-fold ) and lost of activity at the mMC3R compared to the Lysine tetrapeptide 68. This striking contrast be tween the carbamate and tetrapeptide derivatives i llustrates the importance of an appropriate pharmacophore extending from the side chain of the tetrapep tide for selectivity and efficacy at the melanocortin receptors. The -amino group of the lysine in tetrapeptide 68 was reacted with various isocyanates and isothiocyanates to explor e which functional group (urea or thiourea) might suffice. Nine analogues, 74-82 (Table 6-2 and Figure 6-2) bearing either an alkyl or aryl urea or thiourea were synthesized and compared to determine which functionality would be better for further development. Conve rsion of urea to thi ourea in this series showed no difference in potency at the mMC3 -5 receptors. There was also no significant difference in the analogues with alkyl (modified peptide 74, 75, 80, 81 , and 82 ), cyclo alkyl (modified peptide 78 and 79 ) or aryl (modified peptide 76 and 77 ) urea or thiourea agonist activity at the MC3-5R. All nine Arg modified tetrapeptide (74-82 ) resulted in decrease potency compared to 67 in the range of 30to 760fold. Due to the lack of

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108 differences in potency among these anal ogues, tetrapeptide derivatives bearing substituted ureas in the side chain were further developed. The phenylurea, 76, with equipotent a gonist activity at the mMC3R and 18-fold and 6-fold decreased activity at the mM C4R and mMC5R respectively, compared to 68 was studied further (Table 6-2 and Figure 63). Systematic substitution about the phenyl ring with alkyl ( 85, 92 ), phenyl ( 89 ), chloro ( 83-84 ), ether ( 86, 90 ), nitro ( 91 ) and acetyl ( 93 ) demonstrated that the 4-substituted an alogues were generally more potent at the mMC1R. 67 68 74 75 76 77 78 79 80 81 82 mMC1R mMC5R mMC4R mMC3R 0 20000 40000 60000 80000 100000 R = X = S O O S O SO S S Peptide 74 75 76 7778798081 82 HN O HN O NH O N HN HN NH XR HN O H2N O NHEC50(nM)>NA 67 68 74 75 76 77 78 79 80 81 82 mMC1R mMC5R mMC4R mMC3R 0 20000 40000 60000 80000 100000 R = X = S O O S O SO S S Peptide 74 75 76 7778798081 82 HN O HN O NH O N HN HN NH XR HN O H2N O NHEC50(nM)>NA Figure 6-2. Comparison of agonist activity at the mouse mela nocortin receptors for urea and thiourea analogues modified at th e Lys side-chain of Ac-His-DPhe-LysTrp-NH2 tetrapeptide The 4-methyl (85), and 4-chlorophenyl (84) derivatives improved mMC5 receptor agonist activity by roughly 5-fold over the unsubstituted compound 76. Slight improvement in potency with these two anal ogues was also seen at the mMC1R over the

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109 unsubstituted 76. Each of the phenyl-substitute d analogues had weaker or complete lost of activity for the mMC3 receptor and equi potent activity at the mMC4 receptor when compared to the unsubstituted phenylurea 76. In this series, mMC5R selectivity was achieved with 4-chlorophenyl urea, 84 (roughly 9-fold) and 4-methylphenyl urea, 85 (roughly 7-fold) over the mMC4R and both ha d no agonist activity at the mMC3R. Compound 86 (EC50 = 162 nM) containing the methoxy gr oup at position 4 was selective for the mMC1R over the mMC3R (278-fold), mMC4R (20-fold) and mMC5R (8-fold). Evaluation of the 2,4-dichlorophenyl (94) analogue resulted in an equipotent mMC5R agonist compared to analogue 83 and a 8-fold decrease potency compared to analogue 84 , suggesting that in a given compound cont aining a chloro-substituted phenyl ring, the effects of 2-substitution appear to outweigh the contributions of substituents at the 4-position for mMC5R agonist activity. 67 68 76 83 84 85 86 90 91 92 93 94 mMC1R mMC5R mMC4R mMC3R 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000N A E C5 0( n M ) HN O HN O NH O N HN HN NH OR HN O H2N O NH Cl76 8384 8586 90 9192 93Cl OCH3OC2H5NO2O Cl Cl94 R = 67 68 76 83 84 85 86 90 91 92 93 94 mMC1R mMC5R mMC4R mMC3R 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000N A E C5 0( n M ) HN O HN O NH O N HN HN NH OR HN O H2N O NH Cl76 8384 8586 90 9192 93Cl OCH3OC2H5NO2O Cl Cl94 R = Figure 6-3. Comparison of agonist activity at the mouse melanocortin receptors for phenyl-substituted urea modified at the Lys side-chain of Ac-His-DPhe-LysTrp-NH2 tetrapeptide Replacement of the phenyl group with 1and 2-naphthylene produced ureas 87 and 88, respectively (Table 6-2). Th e compounds were equipotent for the mMC5R, lost all

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110 agonist activity at the mMC3R and the 2-naphthyl derivative 88 was approximately 5-fold more selective for the mMC4 R over the 1-naphthyl derivative 87. The presence of an aromatic urea is not mandatory for mMC4 and mMC5 receptors activity, since the cyclohexyl urea 78 was equipotent with its unsaturated counterpart 76. The monosubstituted urea 71 exhibited low agonist activity for the mMC5 when compared to most of the disubstituted examples, indicating that the presence of an N,N’-disubstituted urea appears to be critical for impr ove potency at the mMC5R receptor. Comparison of benzylurea 75 to 96-97 with methyl substituted at the position were prepared and tested for melanocortin receptor agonist ac tivity and selectivity (Table 6-2). The benzyl derivative 75 exhibited comparable properties to the R-methyl (96) and S-methyl (97) analogues at the mMC3R. All three derivatives maintained agonist activities at the mMC3-5R but analogue 97 was selective for the mMC1R (EC50 = 309 nM) with 40-fold selectivity over the mMC4R, 125-fold over the mMC3R and 11-fold over the mMC5R. Although the R-methyl derivative 97 (EC50 = 768 nM) was as equipotent (within experimental error) as the S-methyl derivative 96 at the mMC1R, it was only 13-fold selective over the mMC4R, 55-fold over the mMC3R and 7-fold over the mMC5R. The results suggest that stereochemistry of the substituent at the -methyl position plays an important role for mMC1R sel ectivity and potency in this series of urea analogues. Summary We have designed, synthesized and characte rized a tetrapeptide library of 31 me mbers, as discussed above. The peptid es were each purified to homogeneity and verified for structural integr ity by various analytical means. The peptides were analyzed

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111 for agonist functional activit y at the cloned mouse MC3-MC5 receptors to identify structure-activity relationship trends, recepto r selectivity character istics, and with the hope of identifying compounds that exhibi ted novel and useful pharmacology. The information obtained is important in the de velopment of lead co mpounds for utilization in the rational drug design process and also to provide insights into the types of interactions that are required for mol ecular recognition and stimulation of the melanocortin receptor isoforms. The data suppor ted the hypothesis that the Arginine side chain in Ac-His-DPhe-Arg-Trp-NH2 tetrapeptide is important for ligand potency at the mouse MC3 and MC4 receptors. However, Argi nine is not essential for melanocortin receptor agonist activity since most of the modi fied tetrapeptide with side chain urea or thiourea had agonist activity. We were able to identify a novel mMC1R selective agonist Ac-His-DPhe-Lys(phenyl thiourea)-Trp-NH2 (EC50 = 36 nM), compound 77 with 703-, 160-, and 21-fold selectivity over the mMC3R, mMC4R and mMC5R respectively (Figure 6-4). As discussed in this chapter, we have identified several modifications that can be incorporated at the arginine positi on in the core His-Phe-Arg-Trp sequence to result in functional activity that is both novel and synthetically useful. 0 5000 10000 15000 20000 25000 30000 mMC1RmMC5RmMC4RmMC3R HN HN O NH O HN HN O NH2O H3C O NH NH S N HNEC50(nM) 703-fold 160-fold 21-fold 0 5000 10000 15000 20000 25000 30000 mMC1RmMC5RmMC4RmMC3R HN HN O NH O HN HN O NH2O H3C O NH NH S N HNEC50(nM) 703-fold 160-fold 21-fold Figure 6-4. Illustration of Ac-His -DPhe-Lys(phenyl thiourea)-Trp-NH2 modified tetrapeptide a mMC1R selective agonist

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112 CHAPTER 7 EXPERIMENTAL Peptide Synthesis AGRP Elongation Peptides Peptide synthesis was performed usi ng standard 9-fluorenylmethoxycarbonyl (Fmoc) methodology in a manual reaction ve ssel or by semi-automation on Advanced Chemtech labtech. The amino acids Fmoc-Le u, Fmoc-Lys(Boc), Fmoc-Arg(Pbf), Fmoc-Ala, Fmoc-Tyr(tBu), Fmoc-Cys(Acm), Fm oc-Phe, Fmoc-Asn(Trt), Fmoc-Thr(tBu), Fmoc-Pro, Fmoc-Asp(OtBu), Fmoc-Val, Fmoc-G ln(Trt), and Fmoc-Gly were purchased from Peptides International (Louisville, KY, USA). The peptides were assembled on 9-fluorenylmethoxycarbonyl-L-leucine-p-alkoxyb enzyl alcohol Fmoc-Leu Wang) resin (0.73 meq/g substitution) or Rink-amidep-methylbenzylhydrylamine (Rink-amide-MBHA) resin (0.40 meq/g substitution) purchased from Peptides International. All reagents were ACS grade or better. The Fm oc protecting groups were removed using 20% piperidine (Sigma Aldric h) in N,N-dimethylformamid e (DMF), amino acid coupling (3-fold excess) was accomplished using 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU, 3-fold excess), 1-hydroxybenzotriazole anhydrous (HOBt, 3-fo ld excess) and diisopropylethylamine (DIEA, 5.1-fold excess). Completion of ami no acid coupling and Fmoc deprotection were monitored using the ninhydrin test.103 Final peptide cleavage from the resin and amino acid side chain protecting group removal was performed using (82.5:5:5:5:2.5)

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113 trifluoroacetic acid (TFA), phenol, water, thio anisole, and 1,2-ethanedithiol or (88:5:5:2) TFA, phenol, water and triethylsilane for 23 hours. Peptide cycliz ation was performed by either of two methods, A or B below. Reversed-phase high performance liquid chromatography (RP-HPLC) purification was performed using a Shimadzu chromatography system with a photodiode arra y detector. Final pept ide purification was achieved using a semi-preparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm). The purified peptides were >96% pure as determined by analytical RP-HPLC and had the correct mol ecular mass (University of Florida protein core facility). Method A: cyclization in solution183 The linear peptides were cleaved from the resin using (82.5:5:5:5:2.5) TFA:phenol: water:thioanisole:1,2-ethanedithiol for 3 h at room temperature (RT). The crude linear peptides, the bis(Acm) intermediates, were dissolved in glacial acetic acid – water (gHOAc-H2O; 4:1) and iodine (10 eq) dissolved in methanol was then added. Cyclization was monitored by RP-HPLC and mass spectrosc opy. The reaction was stirred in the dark at RT for 90 min, quenched by diluting with H2O (twice the amount of the total volume used for cyclization) and extrac ted with carbon tetrachloride (CCl4; 4 x 15 m L ) to remove excess iodine. The aqueous phase was then lyop hilized to give the crude cyclic peptide. Method B: cyclization on resin183 In a manual reaction vessel, the synthesized linear peptide attached to the resin was wash with DMF (4 x 10 mL). Iodine (15 eq ) dissolved in DMF was added to the resin and mixed in the dark with nitrogen gas for 2 h at RT. The peptide resin was washed with DMF, dichloromethane (DCM) and 1,2-dichlo roethane (10 x 2 min, 10 mL) followed by

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114 DCM (5 x 2 min, 10 mL ). Cleavage of the cyclic peptides from the resin was achieved with (88:5:5:2) TFA:phenol:water: triethylsilane for 2 h at RT. Figure 7-1. Picture of the Manual reaction vessel used in AGRP elongation, urea and modified tetrapeptide studies AGRP Stereochemistry Peptides Peptide synthesis was performed usi ng standard 9-fluorenylmethoxycarbonyl (Fmoc) methodology93,94,184 by automation on an Advanced ChemTech 440MOS automated synthesizer (Louisville, KY, USA) . The amino acids Fmoc-Lys(Boc), FmocArg(Pbf), Fmoc-DArg(Pbf), Fmoc-Ala, Fm oc-Tyr(tBu), Fmoc-Cys(Acm), Fmoc-Phe, Fmoc-DPhe, Fmoc-Asn(Trt), Fmoc-Thr(tBu ), Fmoc-Pro, Fmoc-Asp(OtBu), and FmocVal were purchased from Peptides Interna tional (Louisville, KY, USA). The peptides were assembled on 9-fluorenylmethoxycar bonyl-L-leucine-p-al koxybenzyl alcohol (Fmoc-Leu Wang) resin (0.73 meq/g substitution ), purchased from Pe ptides International (Louisville, KY, USA). All reag ents were ACS grade or bett er and were used without

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115 further purification. The Fmoc protecting gr oups were removed us ing 20% piperidine (Sigma Aldrich) in N,N-dimethylformamide (DMF), amino acid coup ling (3-fold excess) was accomplished using 2-(1H-benzot riazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 3-fold excess) , 1-hydroxybenzotriazole anhydrous (HOBt, 3-fold excess) and diisopropylethylamine (DIEA, 5.1-fold excess) manually. On the automated synthesizer the synthesis was perf ormed using a 16 well teflon reaction block with a course frit. Approximately 274 mg re sin (0.2 mmole) was added to each reaction block well. The resin was allowed to swell for 2 h in methylene chloride (DCM) and deprotected using 25% piperi dine in DMF twice for 5 min then 20 min at 450 rpm. A positive ninhydrin test103 result indicates free amine gr oups on the resin. The growing peptide chain was added to the Wang resin us ing the following general amino acid cycle: 500 L DMF is added to each reaction well to wet the frit, 3-fold excess amino acid starting from the C-terminus is added (900 L of 0.5M amino acid solution containing 0.5M HOBt in DMF) followed by the additi on of 900 L 0.5M diisopropylcarbodiimide (DIC) in DMF and the reaction well volum e is brought up to 6 ml using DMF. The coupling reaction is mixed for 1 h at 450 rpm, followed by emptying of the reaction block by positive nitrogen gas pressure. A second coup ling reaction is performed by addition of 500 L of DMF to each reaction vessel, follo wed by addition of 900 L of 0.5 M of the respective amino acid, 900 L 0.5 M HBTU, 765 L 1M DIEA, the reaction well volume is brought up to 6 m L with DMF and mixed at 450 rpm for 1 h. After the second coupling cycle, the reaction block is emptied and the Fm oc protected resin is washed with DMF (6 mL 4 times). Fmoc deprotection is performed by addition of 6 mL 25% piperidine in DMF and for 5 min at 450 rpm followed by a 20 min deprotection at 450 rpm. The reaction

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116 well is washed with 6 mL DMF 4 times and the next coupling cycle is performed as above. Completion of amino acid coupling and Fmoc deprotection were monitored using the ninhydrin test.103 Final peptide cleavage from the resin and amino acid side chain protecting group removal was performed using 5 ml of (82.5:5:5:5:2.5) trifluoroacetic acid (TFA), phenol, water, thioanisole, and 1,2ethanedithiol or (88: 5:5:2) TFA, phenol, water and triethylsilane for 2-3 hours. Pep tide disulfide cyclization was performed by either of two methods, A or B below. The cleavage product was emptied from the reaction block into a cleavage block containing 15 mL colle ction vials under nitrogen gas pressure. The resin was washed with 3 ml cleavage cocktail for 5 min at 450 rpm and emptied into the previous cleavage solution. The crude peptides was transferred to preweighed 50 mL conical tubes and precipitated with cold (4 C) anhydrous ethyl ether (up to 50 mL ). The crude peptides was centrif uged (Sorval Super T21 high speed centrifuge using the swing bucket rotor) at 4000 rpm for 5 min and 4 C. The ether was decanted off and the peptide was washed one more time w ith cold anhydrous ethyl ether and pelleted as before. The crude peptides were dried in vacuo for 48 h. The crude peptide yields ranged from 75 to 95% of the theoretical yields based on resin loading. A 30 to 40 mg sample of crude peptide was purified by Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) using a Shim adzu chromatography system with a photodiode array detector. Final peptide purification was achie ved using a semipreparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm). The purified peptides were >96% pure as determ ined by analytical RP-HPLC in two diverse solvent systems and had the correct molecula r mass (University of Florida protein core facility).

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117 Figure 7-2. Picture of the Advanced Chemt ech 440 MOS synthesizer used to prepare MSH and AGRP stereochemical analogues Method A: disulfide bridge formation in solution183 The linear peptides were cleaved from the resin using (82.5:5:5:5:2.5) TFA:phenol: water:thioanisole:1,2-ethanedithiol for 3 h at room temperature (RT). The crude linear peptides, the bis(Acm) intermediates, were dissolved in glacial acetic acid – water (HOAc-H2O; 4:1) and 10 eq iodine dissolved in methanol was added. Cyclization was monitored by RP-HPLC and mass spectroscopy. The reaction was mixed in the dark at RT for 90 min, quenched by diluting with wate r (twice the amount of the total volume used for cyclization) and extrac ted with carbon tetrachloride (CCl4; 4 x 15 m L ) to remove excess iodine. The aqueous phase was then lyop hilized to give the crude cyclic peptide. Method B: disulfide bridge formation on resin 183 In a manual reaction vessel, the synthesized linear peptide attach to the resin was washed with DMF (4 x 10 m L ). Iodine (15 e q) dissolved in DMF wa s added to the resin and mixed in the dark by bubbling with nitroge n gas for 2 h at RT. The peptide resin was

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118 washed with DMF, dichloromethane (DCM) and 1,2-dichloroethane (10 x 2 min, 10 m L ) followed by DCM (5 x 2 min, 10 mL ). Cleavage of the cy clic peptides from the resin was achieved with (88:5:5:2) TFA:phenol:wat er: triethylsilane for 2 h at RT. -MSH Peptides Peptide synthesis was performed usi ng standard 9-fluorenylmethoxycarbonyl (Fmoc) methodology93,94,184 by automation on an Advanced ChemTech 440MOS automated synthesizer (Louisville, KY, USA) . The amino acids Fmoc-Tyr(tBu), Fmoc-Val, Fmoc-Met, Fmoc-Gly, Fmoc-His(Trt), Fmoc-Phe, Fmoc-Arg(Pbf ), Fmoc-Trp(Boc), and Fmoc-Asp(OtBu) were purchased from Peptides International (Louisville, KY). The peptides were assembled on 9-fluorenylmethoxycarbonylL-amino acid-p-alkoxybenzyl alcohol (Fmoc-amino acid Wang) resin (0.40 to 0.62 meq/g substitution), purchased from Peptides International (Loui sville, KY). All reagents were ACS grade or better and were used without fu rther purification. The Fmoc protecting groups were removed using 20% piperidine (Sigma Aldrich) in N,N-dimethylformamide (DMF), amino acid coupling (3-fold excess) was acco mplished using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophospha te (HBTU, 3-fold excess), 1-hydroxybenzotriazole anhydrous (HOBt, 3-fo ld excess), and diisopropylethylamine (DIEA, 5.1-fold excess) manually. On the automated synthesizer, the synthesis was performed using a 16-well teflon reaction block with a coarse fit. Approximately 250 mg resin (0.2 mmole) was added to each reaction block well. The resin was allowed to swell for 2 h in methylene chloride (DCM) and was deprotected using 25% piperidine in DMF twice (for 5 min, then 20 min) at 450 rpm. A positive ninhydrin test103 result indicates free amine groups on the resin. The growing peptid e chain was added to the Wang resin using the following general amino acid cycle: 500 L DMF is added to each reaction well to wet

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119 the frit, 3-fold excess amino acid starting from the C-termi nus is added (900 µl of 0.5M amino acid solution containing 0.5M HOBt in DMF) followed by the addition of 900 µl 0.5M diisopropylcarbodiimide (DIC) in DMF and the reaction well volume is brought up to 6 ml using DMF. The coupling reaction is mixed for 1 h at 450 rpm, followed by emptying of the reaction block by positive ni trogen gas pressure. A second coupling reaction is performed by addition of 500 µl of DMF to each reaction vessel, followed by addition of 900 µl of 0.5 M of the respectiv e amino acid, 900 µl 0.5 M HBTU, 765 µl 1M DIEA, the reaction well volume is brought up to 6 ml with DMF and mixed at 450 rpm for 1 h. After the second coupling cycle, th e reaction block is emptied and the Fmoc protected resin is washed with DMF (6 ml 4 times). Fmoc deprotection is performed by addition of 6 ml 25% piperidi ne in DMF and for 5 min at 450 rpm followed by a 20 min deprotection at 450 rpm. The reaction well is washed with 6 ml DMF 4 times and the next coupling cycle is perfor med as above. Completion of amino acid coupling and Fmoc deprotection were monitore d using the ninhydrin test.103 Final peptide cleavage from the resin and amino acid side ch ain protecting group removal wa s performed using 5 ml of (89.9:5:5:0.1) trifluoroacet ic acid (TFA), triethylsilane, water, and p -thiocresol/ p -cresol (1:1) for 2-3 h. The cleavage product was emptie d from the reaction block into a cleavage block containing 15 ml collection vials unde r nitrogen gas pressure. The resin was washed with 3 ml cleavage cocktail for 5 mi n at 450 rpm and emptied into the previous cleavage solution. The crude peptides was tran sferred to pre-weighed 50 ml conical tubes and precipitated with cold (4 C) anhydrous ethyl ether (up to 50 ml). The crude peptides was centrifuged (Sorval Super T21 high speed cen trifuge using the swi ng bucket rotor) at 4000 rpm for 5 min and 4 C. The ether was decanted off and the peptide was washed one

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120 more time with cold anhydrous ethyl ether and pelleted as before. The crude peptides were dried in vacuo for 48 h. The crude pep tide yields ranged from 75% to 95% of the theoretical yields based on resin loading. A 30 to 40 mg sample of crude peptide was purified by Reversed-Phase High Perform ance Liquid Chromatography (RP-HPLC) using a Shimadzu chromatography system with a photodiode array detector. Final peptide purification was achieved using a semi-preparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm). The purified pep tides were >96% pure as determined by analytical RP-HPLC in two diverse solv ent systems and had the correct molecular mass (University of Florida protein core facility). Urea Analogues The solid-phase synthesis of the library compounds used in this study is illustrated in figure 7-3. The chemistry has been m odified from previously published methods.165,166 The diamine, R1 subunit was coupled to 4-[4-(hydroxymethyl)-3methoxyphenoxy]butanoyl-p-methylbenzhydrylam ine (HMPB-MBHA) resin through a carbamate linkage after activation of the re sin with p-nitrophenylchloroformate. The Fmoc-protected amino acid, R2 subunit was then added to th e amine to form an amide bound with diisopropylcarbodiim ide (DIC) and 3 % N,N-(d imethylamino)-pyridine (DMAP) in N,N-dimethylformamide (DMF) as the coupling reagents. This was followed by Fmoc deprotection with 20% piperidine. The R3, amine subunit was subsequently attached through a urea linka ge after carbonylation of the free amine of the R2 subunit. The product was cleaved from the re sin with glacial acetic acid at 45 û C on an Advanced Chemtech 440MOS automated synthesizer. Compound synthesis was performed using a manual reaction vessel or by semi-automation on an Advanced Chemtech LabTech. The assayed compounds were >96% pure as dete rmined by analytical RP-HPLC in two

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121 solvent systems, had the correct molecula r mass as determined by mass spectrometry (University of Florida protein co re facility). One dimensional 1H NMR (Brucker Advance 500, d-MeOH, Appendix A) was used to verify th at the correct chemical structures were obtained (University of Florid a McKnight Brain Institute). H N O O OCH3OH H N O O OCH3ON H O R1N H O NH2R2H N O O OCH3ON H O R1N H O H N R2NHR3O O NHFmoc R2H N O O OCH3ON H O R1N H H2N R1N H O H N R2NHR3O R1H N O O OCH3ON H O NH2C3H7HNNHC3H7O NO2HO++ Reagents and conditions: (a) p-nitrophenylchloroformate (18.7 eq), diisopropylethylamine (DIEA, 18.7 eq), THF/CH2Cl2 (1:1), overnight; ( b) diamine, R1 (12.5 eq), DIEA (12.5 eq), DMF, overnight; (c) Fmoc-amino acid, R2 (7.2 eq), DIC (7.2 eq), DMAP (0.22 eq), DMF, 2 x 3 hr; (d) 20 % piperidine in DMF, 30 min; (e) p-nitrophenylchloroformate (1.4 eq), diisopropylethylamine (DIEA, 1.4 eq), THF/CH2Cl2 (1:1), 1 hr; (f) amine, R3 (1.4 eq), DIEA (1.4 eq) DMF, overnight; (g) Acetic acid , 45 oC, 24 hr. g a, b c d e, f Figure 7-3. Schematic representation for urea synthesis using solid phase methodology Modified Tetrapeptide Peptide synthesis will be performed using standard 9-fluorenylmethoxycarbonyl (Fmoc) methodology in a manual reaction vessel or by automation on Advanced Chemtech 440MOS. The amino acids Fmoc-Lys (Z), Fmoc-Lys(Aloc), Fmoc-Arg(Pbf), Fmoc-DPhe, Fmoc-His(Trt), and Fmoc-T rp(Boc) will purchased from Peptides International (Louisville, KY, USA), Bachem or Advanced Chemtech. The peptides were assembled on Rink-amide-p-methylbenzylhydr ylamine (Rink-amide-MBHA) resin (0.40

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122 meq/g substitution), purchased from Peptides In ternational. All reagents were ACS grade or better. The Fmoc protecting groups will be removed using 20% piperidine (Sigma Aldrich) in N,N-dimethylformamide (DMF), amino acid coupling (3-fold excess) was accomplished using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 3-fold excess) , 1-hydroxybenzotriazole anhydrous (HOBt, 3-fold excess) and diisopropylethylamine (D IEA, 5.1-fold excess). Completion of amino acid coupling and Fmoc deprotection will be monitored using the ninhydrin test. Final peptide cleavage from the re sin and amino acid side chain protecting group removal was performed using (95:2.5:2.5) trif luoroacetic acid (TFA), wate r, and triisopropylsilane for 2-3 hours. Reversed-phase high performance liquid chromatography (RP-HPLC) purification will be performed using a Shimadzu ch romatography system with a photodiode array detector. Final peptide puri fication will be achieved using a semipreparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 x 25 cm). Removal of aloc protecting group The resin-bound linear tetrapep tide was dried completely in vacuo and then swollen with chloroform (CHCl3) under an atmosphere of Argon. To this was added one-half of a mixture of palladium tetrak is(triphenylphosphine) [Pd(PPh3)4] (3 eq based on resin loading), acetic acid (2.5%), and N -methylmorpholine (5%) in CHCl3. After stirring at room temperature (RT) for 4 h, the palladium (Pd) solution will be drained, and the resin will be treated with the second half of the Pd mixture and allowed to stir at RT for 12 h. (Figure 7-4) The side chain deprotected resin product was filtered and washed with dichloromethane (DCM).

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123 Preparation of ureaor thiourea-substituted tetrapeptides A solution of the tetrapeptide, isocyana te or isothiocyanate (5.0 eq), and N methylmorpholine (7.0 eq) in DMF was stirred at room temperature for 18 h. (Figure 74). The resin was filtered and washed with DMF followed by DCM. The resin was then cleaved provide the crude final product. HN HN O NH O HN HN O NH O H3C O NH O O N HN HN HN O NH O H2N HN O NH O H3C O NH N HN HN HN O NH O HN HN O NH2O H3C O NH NH XR N HN HN HN O NH O HN HN O NH O H3C O NH NH XR N HN 3-6 eq Pd(PPh3)4, in chloroform containing 2.5% acetic acid & 5% N-methylmorpholine. Mix at rt for 4 h with half the mixture, then filter and repeat with the 2nd half of reagent for 12 h Filter and wash resin with DCM 5.0 eq isocyanate (RNCO) or isothiocyanate (RNCS) and 6.2 eq N-methylmorpholine in DMF at room temperature for 18 h TFA/H2O/triisopropylsilane (95:2.5:2.5), 2 h at room temperatureX = O, S R = R group of isocyanate or isothiocynate Figure 7-4. Schematic representation for si de chain modification using solid phase methodology Cell Culture and Transfection Briefly, HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), 10% pen-strep, with 10% fetal calf serum (FCS) and seeded 1 day prior to transfection at 1¯2 x 106 cells/100-mm dish. Mouse mela nocortin receptor cDNA (20 µg) in the pCDNA3 expression vector were transfec ted by using the calcium phosphate

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124 method. Stable receptor populations were gene rated by using G418 sulfate selection (1 mg/ml) for subsequent bioassay analysis.122 Figure 7-5. Picture of the semi-automated Advanced ChemTech synthesizer used to prepare urea and modified tetrapeptide analogues -Galactosidase Functional Bioassay HEK-293 cells stably expressing the melanoc ortin receptors were transfected with 4 µg CRE/ -galactosidase reporter gene as previously described.185 Briefly, 5,000-15,000 post-transfection cells were plated into 96-we ll Primera plates (Falcon) and incubated overnight. Forty-eight hours post-transfection, th e cells were stimulated with peptide (104 to 10-12 M or forskolin control (10-4 M) in assay medium (DMEM containing 0.1 mg/ml BSA and 0.1 mM isobutylmethylxanthi ne, IBMX) for 6 h. The assay media was aspirated and 50 µl of lysi s buffer (250 mM Tris·HCl, pH 8.0, and 0.1% Triton X-100) was added. The plates were stored at -80° C overnight. The plates containing the cells lysates were thawed the following day. Aliquot s of 10 µl were taken from each well and transferred to another 96-well plate for rela tive protein determina tion. Phosphate-buffered

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125 saline (PBS) with 0.5% BSA (40 µl) was adde d to each well of the cell lysate plates. Subsequently, 150 µl substrate buffer (60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM -mercaptoethanol, 200 mg/ml ONPG) wa s added to each well, and the plates were incubated at 37°C. The samp le absorbance, OD405, was measured using a 96-well plate reader (Molecular Devices). The relative protein value was determined by adding 200 µl 1:5 dilution Bio-Rad G250 prot ein dye:water to th e 10-µl cell lysate sample taken previously, and the OD595 was measured on a 96-well plate reader (Molecular Devices). Forskolin treatment (10-4 M) in six we lls of each 96-well plate was used as controls for transfection efficiency of the CRE-gal reporter gene. Data points were normalized both to the re lative protein content and nonreceptor-dependent forskolin stimulation. Data analysis and EC50 values were determined by using nonlinear regression analysis with the PRISM program (v3.0, Gra ph Pad Inc.). The antagonistic properties were determined by the ability of these ligands to competitively displace the agonist MTII in a dose-dependent manner. The pA2 values were generated using the Schild analysis method,186 as previously reported.187,188 The pA2 values were then used to calculate the corresponding Ki values (Ki = -log pA2). The EC50 and Ki values represent the mean of duplicate wells examined in at l east three independent experiments, with the standard errors of the mean presented. NDP-MSH and AGRP(86-132) Iodination 125I-NDP-MSH and 125I-AGRP(86-132) were prep ared using a modified chloramines-T method as previ ously described by Yang, et.al.113 Using 50 mM sodium phosphate buffer, pH 7.4 as the reaction buffer, 125I-Na (0.5 mCi, Amersham Life Sciences, Inc., Arlington Heights, IL) wa s added to 20 µg of NDP-MSH (Bachem,

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126 Torrance, CA) or 20 µg of hAGRP(86-132) (Pep tide International, Inc., Osaka Japan) in 5 µl buffer. To initiate the reaction, 10 µl of a 2.4 mg/ml solution of chloramines-T (Sigma Chemical Co., St. Louis, MO) was added for 15 s with gentle agitation. This reaction was terminated by the addition of 50 µl of 4.8 mg/ml solution of sodium metabisulfate (Sigma Chemical Co.) for 20 s with gentle agitation. The reaction mixture was then diluted with 200 µl 10% bovine serum albumin and the resultant mixture layered on a Bio-Gel P6 (Bio-Rad Labs, Hercules, CA) column (1.0 x 30 cm Econocolumn, Bio-Rad Labs) with hAGRP a nd on a Bio-Gel P2 (Bio-Rad Labs, Hercules, CA) column (1.0 x 50 cm Econoc olumn, Bio-Rad Labs) with NDP-MSH for separation by size exclusion chromatography using 50 mM sodium phosphate buffer, pH 7.4 as column eluant. Fifteen drop fractions (approximately 500 µl) were collected into glass tubes containing 500 µl of 1 % BSA. Each fraction was then counted on the Apex Automatic Gamma Counter (ICN Micromed ic Systems Model 28023, Huntsville, AL with RIA AID software, Robert Maciel Asso ciates, Inc., Arlington. MA) to determined peak 125I incorporation fractions. Receptor Binding Studies of AGRP Elongation Analogues HEK-293 cells stably expressing the melanoc ortin receptors were maintained as described above. One day preceding the experiment, 0.3 × 106 cells/well was plated into Primera 24 well plates (Falcon). The peptid es NDP-MSH, hAGRP(86-132) and peptides 1-8 were used to competitively displace the I125-radiolabeled NDP-MSH or hAGRP(86132) (100,000 cpm/well) in a dose-response (10-5 to 10-12 M) manner. A 450 µl solution of the peptide concentration being tested wa s added to the well. Next, 50 µl solution of I125NDP-MSH or I125hAGRP(86-132) was added to each well, and the cells were incubated at 37°C for 1 h. The medium wa s subsequently removed, and each well was

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127 washed with assay buffer (1 ml, DMEM, 0.1 mg/ml BSA). The cells were lysed by the addition of 0.5 ml 0.1 M NaOH a nd 0.5 ml 1%Triton X-100. The mi xture was left to lyse the cells for 10 min, and the c ontents of each well transferred to labeled 16 × 150-mm glass tubes and quantified using a Titertek 10x600 -counter (ICN Micromedic Systems, Huntsville, AL with RIA AID software, Robe rt Maciel Associates , Inc., Arlington, MA). Dose-response curves and IC50 values were generated and analyzed by nonlinear least squares analysis189 and graphed using prism v3.0 (Graph pad). The IC50 values represent the mean of duplicate wells generated in at least two independent experiments, with the errors presented as standard deviation of the mean. Data Analysis EC50 and pA2 values represent the mean of dup licate experiments performed in quadruplet or more independent experiments. EC50 and pA2 estimates, and their associated standard errors, were determin ed by fitting the data to a nonlinear leastsquares analysis using the PRISM program (v3.0, GraphPad Inc.). Computational Methods for AGRP Monocyclic Modeling The homology model of the hAGRP(87-132)-M elanocortin-4 receptor complex (1) were imported into the Workspace interface of BioMedCAChe® 5.05 software package (CAChe Group, Fujitsu, Portland OR). The model of the 20-residue peptide 5 complexed with receptor was made directly from the hAGRP(87-132) complex model. Residues 87102 and 122-132 of hAGRP(87-132) were delete d and the terminals were capped with charged group. Cys105, Cys108 and Cys119 were replaced by Ala residues and the whole sequence was renumbered. Complex models of 9 and 11 were created by inverting Arg111 or Phe113 of 5 to Dconfiguration and the angles were adjusted to favorable orientations as well as to fit to the correspondi ng binding pockets.

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128 Limited conformation search of bound bicycl ic peptides were carried out within CONFLEX190 module to locate the global minimum. Complex structures of family 135 and 32 were excluded from further analysis b ecause of the steric conflicts found between bicyclic peptides and Melanocortin-4 recep tor. CONFLEX generated a sequence of lowenergy conformers of any shape systemati cally and exhaustively, incorporating downstream, reservoir-filling, corner flap, edge flip, stepwise rotation, and pre-check. All conformations with Boltzmann populations la rger than 1% were subjected to local perturbations and optimized by augmented MM3. All the final structures were analyzed by automatic labeling of hydrogen bonds and bumps in the 3D Structure Window. The calculations were performed on a Dell desktop PC with 1.70 GHz Pentium 4 processor. One-Dimensional 1H Nuclear Magnetic Resonance Spectroscopy Urea Analogues The compounds were analyzed for purity and structural integrity by nuclear magnetic resonance (NMR). The compounds were dissolved in 600 L deuterate methanol (CD3OD). 1H-NMR spectras were obtained at 27C on a Bruker Avance 500 and 600 MHz spectrometer in the Advanced Magnetic Resonance Imaging and Spectroscopy facility at the McKnight Br ain Institute, Univer sity of Florida. 1H data were collected using the decoupler coil of a Bruke r 5 mm TXI probe. The data were processed and analyzed using Bruker XWIN NMR and XWINPLOT software. Arg Modified Ureas, Thioureas, and Carbamates The compounds were analyzed for purity and structural integrity by nuclear magnetic resonance (NMR). The compounds were dissolved in 600 L deuterate dimethyl sulfoxide (DMSOd 6) containing 0.1% trimethylsilane (TMS). 1H NMR spectras were obtained at 27C on a Bruker Avance 500 MHz spectrometer in the Advanced Magnetic

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129 Resonance Imaging and Spectroscopy facility at the McKnight Brain Institute, University of Florida. 1H data was collected using the decoupler coil of a Bruker 5 mm BBO probe. The data were processed and analyzed using Bruker XWINNMR and XWINPLOT software.

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130 CHAPTER 8 CONCLUDING REMARKS Over the last decade genetic and pharm acological evidence has emerged that supports a role for the melanocortin receptor sy stem in weight homeostasis. Current data suggest that the MC3 and MC4 receptors expressed predominan tly in the central nervous system, along with their endogenous agoni sts and antagonist, are key components responsible for the regulation of body weight via the modula tion of both food intake and energy expenditure.191 The studies in this dissertation describe my effort and contribution in the design, synthesis and pharmacological characteriza tion of agonist and antagonist for the melanocortin receptors. A number of other central regulators of food intake and body weight, such as Leptin, Grehlin, peptide YY (PYY) and neuropeptide Y (NPY) receptors have been identified making the search fo r obesity therapeutics more complicated.10,192196 The complex pathways involving these we ight and energy homeostasis regulators along with the melanocortin receptor system mu st be understood to aid in the design of obesity therapeutics. Because body weight is regulated by the br ain, centrally acting agents are necessary to combat obesity. Th e number of CNS targets for new anti-obesity drugs is ever increasing, thanks to the ongoi ng elucidation of mol ecules involved in the communication between peripheral adiposity sign als and central effect or pathways that govern energy homeostasis. Key peripheral signa ls, such as leptin, insulin, and ghrelin, have been linked to the hypothalamic neurope ptide systems, and the networks that integrate these systems have begun to be elucidated.10 The control of body weight is an

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131 extremely complex process, and weight loss is difficult to achieve, thus, it is extremely unlikely that any one approach will prove to be a magic bullet for all obesity. Nevertheless, there is cause for considerable optimism that multiple classes of new antiobesity medications may soon be developed. It is possible that customized cocktails of these agents will enable obesity to be managed. Elongation of hAGRP(109-118) and Stereo chemical Studies of hAGRP(103-122) Studies of AGRP has identifies the co re decapeptide hAGRP(109-118) as the smallest fragment of AGRP needed for antagonist activity at the melanocortin-4 receptor.68,88,125 The decapeptide had no antagonist or agonist activity at the MC3R even though it bind with an IC50 of 9-12 M at the mouse receptor88,125 and 2 M at the human receptor.68 The above information was used as a starting point in an elongation study to identify the amino acid residues required for MC3R antagonism. In the study elongation of the monocyclic decapeptide core hAGRP(109 -118) with extension at the C-terminal, N-terminal or both at the same time was pha rmacologically characterized at the mMC1, mMC3-5 receptors.197 We have synthesized, purified to homoge neity, analytically and pharmacologically characterized over eight anal ogues to identify the amino acid residue(s) required for mMC3R antagonism as discussed in chapter 3. We have identified that extension at the C-terminus was more important for regaini ng MC3R antagonism, especially the arginine at position 120 (AGRP numbering). We have also identified a novel mMC1R agonist template since all eight analogues were full agonist at this rece ptor. The most potent analogue at both the mMC3 & mMC4 recepto rs, hAGRP(103-122) was studied further aimed at improving potency and receptor selectivity.

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132 Mutational analysis of hAGRP identifie d residues Arg-Phe-Phe (111-113) to be critical for binding to both the MC3R and MC4R.68 Single residue substitution of ArgPhe-Phe with alanine in full length hAGRP resulted in approximately 10to 50-fold reduced binding affinity to hMC3R and hMC4R.68 The importance of the Arg-Phe-Phe triplet was also shown by a number of structur e activity studies to be critical for AGRP interaction and recognition of the melanocortin receptors.109,120,122,126,198,199 Human AGRP(103-122) was identified as having 158 nM and 32 nM antagonist activity at the mMC3R and mMC4R, respectiv ely, 600 nM agonist at the mMC-1R and devoid of activity at the mMC5R,125 making it useful for further structure activity studies. The Arg-Phe-Phe residues were replace with their D-stereoisomers either individually, two at a time or all three at once. All of the Derivatives except the one containing DPhe112 lost antagonist activity at both the mMC3 and mMC4 receptors and all of them became full M agonist at the mMC5R. Interest ly, all of the ligands became full M or partial agonist at both the mMC3 and mMC4 receptors.200 It can be deduced from the results obtained that both the na turally L-configuration of Arg111 and Phe113 are required to maintained mMC3R and mMC4R antagonism. To understand the conversion from anta gonist to agonist the following three ligands, hAGRP(103-122), hAGRP(103-122, Arg111DArg), and hAGRP(103-122, Phe113DPhe), were docked into the mMC-4R. Th e modeling data showed the positively charged DArg111 interacting with Asn115 in TM3 of the mMC-4R which was not seen with Arg111,200 and this interaction may be the reas on for the conversion. In the case of DPhe113 it was shown to be intera cting in a hydrophobic bindi ng pocket identified to be important for antagonism and molecular recognition.122 However, the only differences are

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133 that DPhe113 has a much stronger inte raction with Phe176 of mM C-4R compared to Phe at position 113,200 and may be a possible explanation as to why the DPhe113 analog became an mMC4R agonist. Truncation of 2-MSH In our efforts to understand how the MC 3R is involved in weight and energy homeostasis, truncation st udies were carried out on 2-MSH. It has been determined that 2-MSH can be used to distingu ish the MC3R from the other subtypes due to its moderate selectivity of 7 to 120-fold with an average EC50 of approximately 6 nM at the human melanocortin receptors.27,131,132,139,170,201 Our initial SAR study of 2-MSH, involve peptide truncation to determine the minimum sequence necessary for bioactivity. In an extensive study presented in chapter four of this dissertation, truncation of 2-MSH were performed with removal of one amino acid at a time from either the Cor N-terminal or both at the same time. The analogues were pharmacologically evaluated at the mouse MC1R, MC3R-MC5R. The study identified a ten amino acid fragment as the minimal sequence without lost of potency at the mouse melanocortin receptors. Our results identified 2-MSH as being equipotent at the mM C3 and mMC5 receptors which is a contradiction to the reported data that 2-MSH is slightly selec tive for the MC3R over the MC4R and MC5R.27,131,132 The difference may be due to difference in species used for pharmacological characterization and is therefor e valuable information that needs to be considered when designing lig ands. The two smallest fragme nts, each with six amino acids with melanocortin receptor agonist activity, both had Phe6, Arg7 and Trp8 ( 2-MSH numbering) which is common to all the e ndogenous melanocortin ligands. Hence, these residues are very important for agonist activity at the melanocortin receptors.

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134 Urea Nonpeptide Analogues In our efforts to develop potent and sele ctive nonpeptides for the melanocortin, we identified a linear urea analogue with 400 nM agonist potency at the mMC4R but it was not selective because it was ju st as potent at the mMC1R.164 However, since the mMC1R is located in the skin and the MC4R is in the brain, it is po ssible to use this analogue as a lead in the development of selective MC4R agonist for the treatmen t of obesity. The aid in the understanding of how the MC3R is involve in energy homeostasis, compound 59 , discussed in chapter five can be used as a starting point in the pr eparation of selective MC3R ligands since this compound had no activity at th e mMC4R. We were able successfully design nonpeptides with improved activity over the starting peptides which is the ultimate goal of research. Structure-Activity Relationship s of Melanocortin Tetrapeptides In our efforts to characterize the melanocor tin system, we recently have carried out truncation studies of NDP-MSH at the clone d central and peripheral mouse melanocortin receptors.122 The Ac-His-DPhe-Arg-Trp-NH2 tetrapeptide was identified as possessing an EC50 value of 10 nM at the murine MC4 receptor, which is similar to the 8 nM results published by Yang et al. for the tetrapeptide at the human MC4 receptor.202 This tetrapeptide exhibits full agoni st efficacy at all the mouse MC receptors and is equipotent to the endogenous hormone -MSH (within experimental error), and only 30-fold less potent than NDP-MSH at the m ouse MC4R. Due to the small size and potency of this peptide, relative to the tridecapeptide -MSH, it was used as a st arting point in a study aimed at improving the potency and receptor se lectivity of the ligand at the melanocortin receptors. The Arg position in this tetrapep tide as been shown to not be required for

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135 agonist activity at the melanocortin receptors but rather for potency.176 In a series of extensive structure-activity relationship (SAR) studies presented in chapter six of this dissertation, modifications we re at the Arg position with the addition of either a carbamate, a urea or a thiourea at the side ch ain to replace the guanidine moiety, with subsequent pharmacological evaluati on at the mouse MC1R, MC3R-MC5R. All of the modifications decreased pote ncy at the melanocortin receptors but a number of them maintained agonist activit y. The study identified peptide 30 as a very selective mMC5R agonist with the methyl group at the -position oriented with an R stereochemistry. This orientation was requi red since the reverse orientation cause a reduction in the selectivity ratio over the othe r receptors even though it was just as potent as peptide 30 at the mMC5R. Although potency was reduced with al l the analogues, we were able to design a novel agonist template for the melanocortin receptors that supports the hypothesis that the guanidine moiety is not required for agonist activity. It would be interested to see whether these analogues bind to the melanocortin receptors to support or disagree with the modeling results that the positive charge on the Arg is needed for interaction with a hydroph ilic pocket in the receptor.153 In conclusion, I think that this disserta tion has illustrated th e success of designing potent and selective ligands fo r the melanocortin receptors as therapeutic agents for the treatment of obesity. Successful in the sense that we were able to develop novel ligand templates as shown when an antagonist be came a melanocortin agonist and these were used to identify residues at the MC4R invol ved in agonist and an tagonist interactions. The results obtained in these studies should aid in the design of melanocortin receptor ligands.

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136 APPENDIX A 1H-NMR FOR NONPEPTIDE UREA ANAL OGUES DISCUSSED IN CHAPTER 5 NH OHN HN O HN H2N C B D E F F G A = 10 aromatic H, 7.06 7.61 B = 2 H, 4.20 4.33 C = 1 H, 4.40 4.45 D = 2 H, 3.05 3.12 E = 2 H, 3.12 3.23 F = 4 H, 1.35 1.54 G = 2 H, 2.73 2.85 A A A A A A A A A A Figure A-1. 1H-NMR of compound 53 discussed in Chapter 5

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137 NH OHN HN O HN H2N C B D D F EA = 10 aromatic H, 6.99 7.65 B = 2 H, 4.21 4.32 C = 1 H, 4.41 4.49 D = 4 H, 3.08 3.25 E = 2 H, 2.71 2.80 F = 2 H, 1.67 1.73A A A A A A A A A A Figure A-2. 1H-NMR of Compound 54 disc ussed in Chapter 5

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138 NH OHN HN O HN H2N E E GA = 10 aromatic H, 6.99 7.62 B = 2 H, 4.23 4.27 C = 1 H, 4.46 4.49 D = 4 H, 3.00 3.25 E = 4 H, 1.30 1.55 F = 2 H, 1.18 1.25 G = 2H, 2.75 2.80C B A A A A A D A A A A A D F Figure A-3. 1H-NMR of compound 55 discussed in Chapter 5

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139 NH OHN HN O HN H2N E E E EA = 10 aromatic H, 6.98 7.62 B = 2 H, 4.24 4.30 C = 1 H, 4.45 4.50 D = 4 H, 2.85 3.25 E = 8 H, 1.15 1.65 F = 2 H, 2.75 2.81C B A A A A A D A A A A A D F Figure A-4. 1H-NMR of compound 56 discussed in Chapter 5

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140 H N O N H N H O H2N NH EA = 14 aromatic H, 6.98 7.62 B = 2 H, 4.24 4.29 C = 1 H, 4.51 4.56 D = 2 H, 3.11 3.27 E = 2 H, 4.30 4.37 F = 2 H, 3.86 3.99C B A A A A A D A A A A F A A A A A Figure A-5. 1H-NMR of compound 57 discussed in Chapter 5

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141 H N O N H N H O H2N NH EA=14a r o m a t ic H, 6.98 7.62 B = 2 H, 4.24 4.29 C = 1 H, 4.51 4.56 D = 2 H, 3.11 3.27 E = 2 H, 4.30 4.37 F = 2 H, 3.86 3.99C B A A A A A D A A A A F A A A A A Figure A-6. 1H-NMR of compound 58 discussed in Chapter 5

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142 N O NH N H O H2N NH E G EA = 10 aromatic H, 6.98 7.61 B = 2 H, 4.23 C = 1 H, 4.45 4.50 D = 2 H, 2.80 3.10 E = 4 H, 3.20 3.35 F = 4 H, 1.05 1.51 G = 1 H, 1.52 1.60 H = 2 H, 2.62 2.72C B A A A A A D F F H A A A A A Figure A-7. 1H-NMR of compound 59 discussed in Chapter 5

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143 NH OHN HN O HN H2N E EA = 10 aromatic H, 7.00 7.61 B = 2 H, 2.65 2.75 C = 6 H, 3.05 3.45 D = 1 H, 4.35 4.45 E = 4 H, 1.35 1.55 F = 2 H, 2.65 2.75D C B C A A A A A C F A A A A A Figure A-8. 1H-NMR of compound 60 discussed in Chapter 5

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144 NH OHN N O HN H2N E G GA = 9 aromatic H, 6.97 7.61 B = 2 H, 4.35 4.51 C = 2 H, 2.82 2.87 D = 2 H, 3.45 3.50 E = 1 H, 4.41 4.46 F = 4 H, 3.11 3.20 G = 4 H, 1.39 1.57 H = 2 H, 2.70 2.80F A A A A A F H D C B A A A A Figure A-9. 1H-NMR of compound 61 discussed in Chapter 5

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145 NH OHN HN O HN H2N N E G JA=10 a r o m a t ic H, 6.96-7.61 B = 2 H, 4.25 4.35 C = 4 H, 2.90 3.10 D = 4 H, 1.50 2.11 E = 1 H, 1.58 1.62 F = 1 H, 4.40 4.49 G = 2 H, 3.52 3.62 H = 2 H, 3.13 3.20 I = 4 H, 1.25 1.50 J = 2 H, 2.71 2.82F A A A A A H I I D C C D B A A A A A Figure A-10. 1H-NMR of compound 62 discussed in Chapter 5

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146 NH OHN N O HN H2N E E G E EA = 10 aromatic H, 6.987.61 B = 2 H, 2.35 2.45 C = 1 H, 1.60 1.80 D = 8 H, 0.80 1.60 E = 8 H, 2.50 2.70 3.05 3.25 3.70 3.90 F = 1 H, 4.35 4.45 G = 2 H, 2.79 2.89F A A A A A D D D C D B A A A A A Figure A-11. 1H-NMR of compound 63 discussed in Chapter 5

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147 H N O N H N H O H2N G G EA = 10 aromatic H, 7.13 7.35 B = 2 H, 4.27 4.37 C = 1 H, 4.06 4.11 D = 2 H, 1.84 2.07 E = 2 H, 2.58 2.75 F = 2 H, 3.19 3.25 G = 4 H, 1.51 1.67 H = 2 H, 2.83 2.93C B A A A A A D F H A A A A A Figure A-12. 1H-NMR of compound 64 discussed in Chapter 5

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148 H N O N H N H O H2N O EA = 14 aromatic H, 6.90 7.51 B = 3 H, 4.19 4.35 C = 6 H, 2.75 3.25 D = 4 H, 1.35 1.65 E = 2 H, 5.10B B A A A A A C C D D C A A A A A A A A A Figure A-13. 1H-NMR of compound 65 discussed in Chapter 5

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149 NH OHN HN O HN H2N E GA = 10 aromatic H, 6.98 7.60 B = 2 H, 4.19 4.31 C = 1 H, 4.40 4.46 D = 2 H, 3.04 3.14 E = 2 H, 3.14 3.23 F = 4 H, 1.34 1.54 G = 2 H, 2.74 2.84C B A A A A A D A A A A A F F Figure A-14. 1H-NMR of compound 66 discussed in Chapter 5

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150 APPENDIX B 1H-NMR OF ARGININE-MODIFIED TETRAP EPTIDES DISCUSSED IN CHAPTER 6 Figure B-1. 1H-NMR of peptide 67 discussed in Chapter 6

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151 Figure B-2. 1H-NMR of peptide 68 discussed in Chapter 6 Figure B-3. 1H-NMR of peptide 69 discussed in Chapter 6

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152 Figure B-4. 1H-NMR of peptide 70 discussed in Chapter 6 Figure B-5. 1H-NMR of peptide 71 discussed in Chapter 6

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153 Figure B-6. 1H-NMR of peptide 72 discussed in Chapter 6 Figure B-7. 1H-NMR of peptide 73 discussed in Chapter 6

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154 Figure B-8. 1H-NMR of peptide 74 discussed in Chapter 6 Figure B-9. 1H-NMR of peptide 75 discussed in Chapter 6

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155 Figure B-10. 1H-NMR of peptide 76 discussed in Chapter 6 Figure B-11. 1H-NMR of peptide 77 discussed in Chapter 6

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156 Figure B-12. 1H-NMR of peptide 78 discussed in Chapter 6 Figure B-13. 1H-NMR of peptide 79 discussed in Chapter 6

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157 Figure B-14. 1H-NMR of peptide 80 discussed in Chapter 6 Figure B-15. 1H-NMR of peptide 81 discussed in Chapter 6

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158 Figure B-16. 1H-NMR of peptide 82 discussed in Chapter 6 Figure B-17. 1H-NMR of peptide 83 discussed in Chapter 6

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159 Figure B-18. 1H-NMR of peptide 84 discussed in Chapter 6 Figure B-19. 1H-NMR of peptide 85 discussed in Chapter 6

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160 Figure B-20. 1H-NMR of peptide 86 discussed in Chapter 6 Figure B-21. 1H-NMR of peptide 87 discussed in Chapter 6

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161 Figure B-22. 1H-NMR of peptide 88 discussed in Chapter 6 Figure B-23. 1H-NMR of peptide 89 discussed in Chapter 6

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162 Figure B-24. 1H-NMR of peptide 90 discussed in Chapter 6 Figure B-25. 1H-NMR of peptide 91 discussed in Chapter 6

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163 Figure B-26. 1H-NMR of peptide 92 discussed in Chapter 6 Figure B-27. 1H-NMR of peptide 93 discussed in Chapter 6

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164 Figure B-28. 1H-NMR of peptide 94 discussed in Chapter 6 Figure B-29. 1H-NMR of peptide 95 discussed in Chapter 6

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165 Figure B-30. 1H-NMR of peptide 96 discussed in Chapter 6 Figure B-31. 1H-NMR of peptide 97 discussed in Chapter 6

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166 LIST OF REFERENCES 1 Center for Disease Control and Preventi on. Prevalence of overweight and obesity among adults: United States, 1985-2002. Available on line http://www.cdc.gov/nccdphp/dnpa/o besity/trend/maps/index.htm . Accessed September 29 2004. 2 Mokdad, A.H., Serdula, M.K., Dietz, W. H., Bowman, B.A., Marks, J.S. and Koplan, J.P. (1999) The Spread of the Obesity Epidemic in the United States, 1991-1998. Journal of the American Medical Association 282 (16), 1519-1522 3 Mokdad, A.H., Ford, E.S., Bowman, B.A., Di etz, W.H., Vinicor, F., Bales, V.S. and Marks, J.S. (2003) Prevalence of Obesity, Diabetes, and Obesity-Related Health Risk Factors, 2001. Journal of the Americ an Medical Association 289 (1), 76-79 4 Mokdad, A.H., Bowman, B.A., Ford, E.S., Vi nicor, F., Marks, J.S. and Koplan, J.P. (2001) The Continuing Epidemics of Obesity and Diabetes in the United States. Journal of the Americ an Medical Association 286 (10), 1195-1200 5 Marti, A., Marcos, A. and Martinez, J. A. (2001) Obesity and immune function relationships. Obesity Reviews 2 (2), 131-140 6 Conway, B. and Rene, A. (2004) Obesity as a disease: no lightweight matter. Obesity Reviews 5 (3), 145-151 7 Lobstein, T., Baur, L. and Uauy, R. ( 2004) Obesity in children and young people: a crisis in public health. Obesity Reviews 5 (s1), 4-85 8 Yang, Y.K. and Harmon, C.M. (2003) R ecent developments in our understanding of melanocortin system in th e regulation of food intake. Obesity Reviews 4 (4), 239-248 9 Finkelstein, E.A., Fiebelkorn, I.C. and Wa ng, G. (2004) State-Level Estimates of Annual Medical Expenditures Attributable to Obesity. Obesity Research 12 (1), 18-24 10 Cummings, D.E. and Schwartz, M.W. (2003) Genetics and pathophysiology of human obesity. Annual Review in Medicine 54, 453-471

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183 peptidomimetic agonists at the human melanocortin receptors MC1R and MC4R. Journal of Medicinal Chemistry 40 (14), 2133-2139 175 Sahm, U.G., Olivier, G.W.J., Branch, S.K., Moss, S.H. and Pouton, C.W. (1994) Influence of [alpha]-MSH terminal ami no acids on binding affinity and biological activity in melanoma cells. Peptides 15 (3), 441-446 176 Holder, J.R., Xiang, Z., Bauzo, R.M. a nd Haskell-Luevano, C. (2003) Structureactivity relationships of the melanocor tin tetrapeptide Ac-His-DPhe-Arg-TrpNH(2) at the mouse melanocor tin receptors. Part 3: modifications at the Arg position. Peptides 24 (1), 73-82 177 Bednarek, M.A., Macneil, T., Kalyani, R. N., Tang, R., Van der Ploeg, L.H.T. and Weinberg, D.H. (1999) Analogs of MT II, Lactam Derivatives of [alpha]Melanotropin, Modified at the N-Termi nus, and Their Selectivity at Human Melanocortin Receptors 3, 4, and 5. Biochemical and Biophysical Research Communications 261 (1), 209-213 178 Bednarek, M.A., Silva, M.V., Arison, B., M acNeil, T., Kalyani, R.N., Huang, R.R.C. and Weinberg, D.H. (1999) Structur e-function studies on the cyclic peptide MT-II, lactam derivative of [alpha]-melanotropin. Peptides 20 (3), 401-409 179 Bednarek, M.A., MacNeil, T., Kalyani, R. N., Tang, R., Van der Ploeg, L.H.T. and Weinberg, D.H. (2000) Analogs of Lactam Derivatives of [alpha]-Melanotropin with Basic and Acidic Residues. Biochemical and Biophysical Research Communications 272 (1), 23-28 180 Cheung, A.W.-H., Danho, W., Swistok, J ., Qi, L., Kurylko, G., Franco, L., Yagaloff, K. and Chen, L. (2002) Structur e-Activity relationship of linear peptide Bu-His-DPhe-Arg-Trp-Gly-NH2 at the hum an melanocortin-1 and -4 receptors: arginine substitution. Bioorganic and Medicinal Chemistry Letters 12 (17), 24072410 181 Schioth, H.B., Mutulis, F., Muceniece, R., Prusis, P. and Wikberg, J.E.S. (1998) Selective properties of Cand N-terminal s and core residues of the melanocytestimulating hormone on binding to the hum an melanocortin receptor subtypes. European Journal of Pharmacology 349 (2-3), 359-366 182 Holder, J.R. and Haskell-Luevano, C. (2004) Melanocortin ligands: 30 years of structure-activity rela tionship (SAR) studies. Medicinal Research Reiewsv 24 (3), 325-356 183 Hargittai, B. and Barany, G. (1999) Contro lled syntheses of natural and disulfidemispaired regioisomers of alpha-conotoxin SI. Journal of Peptide Research 54 (6), 468-479

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184 184 Chang, C.D. and Meienhofer, J. (1978) So lid-phase peptide synthesis using mild base cleavage of N alpha-fluorenylme thyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. International Journal of Peptide and Protein Research 11 (3), 246-249 185 Chen, W.B., Shields, T.S., Stork, P.J.S. and Cone, R.D. (1995) A Colorimetric Assay for Measuring Activation of G( S)-Coupled and G(Q)-Coupled Signaling Pathways. Analytical Biochemistry 226 (2), 349-354 186 Schild, H.O. (1947) Pa, a New Scale fo r the Measurement of Drug Antagonism. British Journal of Pharmacology and Chemotherapy 2 (3), 189-206 187 Haskell-Luevano, C., Lim, S., Yuan, W., Cone, R.D. and Hruby, V.J. (2000) Structure activity studies of the melano cortin antagonist SHU 9119 modified at the 6, 7, 8, and 9 positions. Peptides 21 (1), 49-57 188 Hruby, V.J., Lu, D.S., Sharma, S.D., Castru cci, A.D., Kesterson, R.A., Alobeidi, F.A., Hadley, M.E. and Cone, R.D. ( 1995) Cyclic Lactam Alpha-Melanotropin Analogs of Ac-Nle(4)Cyclo[Asp(5) ,D-Phe(7),Lys(10)] Alpha-MelanocyteStimulating Hormone-(4-10)-Nh2 with Bul ky Aromatic-Amino-Acids at Position7 Show High Antagonist Potency and Se lectivity at Spec ific Melanocortin Receptors. Journal of Medicinal Chemistry 38 (18), 3454-3461 189 Bowen, W.P. and Jerman, J.C. (1995) N onlinear regression using spreadsheets. Trends in Pharmacological Sciences 16 (12), 413-417 190 Goto, H. and Osawa, E. (1993) An E fficient Algorithm for Searching LowEnergy Conformers of Cyclic and Acyclic Molecules. Journal of the Chemical Society-Perkin Transactions 2 (2), 187-198 191 Bednarek, M.A., Hreniuk, D.L., Tan, C., Pa lyha, O.C., MacNeil, D.J., Van der Ploeg, L.H., Howard, A.D. and Feighner, S.D. (2002) Synthesis and biological evaluation in vitro of selective, high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1. Biochemistry 41 (20), 6383-6390 192 Barsh, G.S. and Schwartz, M.W. (2002) Genetic approaches to studying energy balance: perception and integration. Nature Reviews in Genetics 3 (8), 589-600 193 McMinn, J.E., Baskin, D.G. and Schw artz, M.W. (2000) Neuroendocrine mechanisms regulating food intake and body weight. Obesity Reviews 1 (1), 3746 194 Blevins, J.E., Schwartz, M.W. and Baski n, D.G. (2002) Peptide signals regulating food intake and energy homeostasis. Canadian Journal of Physiology and Pharmacology 80 (5), 396-406

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185 195 Morton, G.J. and Schwartz, M.W. (2 001) The NPY/AgRP neuron and energy homeostasis. Intertional Journal Obesity Related Metabolic Disorder 25 Suppl 5, S56-62 196 Baskin, D.G., Blevins, J.E. and Schwar tz, M.W. (2001) How the brain regulates food intake and body weigh t: the role of leptin. Journal of Pediatric Endocrinology and Metabolism 14 Suppl 6, 1417-1429 197 Joseph, C.G., Bauzo, R.M., Xiang, Z., Shaw , A.M., Millard, W.J. and HaskellLuevano, C. (2003) Elongation studies of the human agouti-related protein (AGRP) core decapeptide (Yc[CRFFNAFC]Y) results in antagonism at the mouse melanocortin-3 receptor. Peptides 24 (2), 263-270 198 Wilczynski, A., Wang, X.S., Joseph, C.G., Xiang, Z., Bauzo, R.M., Scott, J.W., Sorensen, N.B., Shaw, A.M., Millard, W. J., Richards, N.G. and Haskell-Luevano, C. (2004) Identification of putative agouti-related pr otein(87-132)-melanocortin-4 receptor interactions by homology mol ecular modeling and validation using chimeric peptide ligands. Journal of Medicinal Chemistry 47 (9), 2194-2207 199 Joseph, C.G., Wilczynski, A., Holder, J.R ., Xiang, Z., Bauzo, R.M., Scott, J.W. and Haskell-Luevano, C. (2003) Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity. Peptides 24 (12), 1899-1908 200 Joseph, C.G.W., Xiang S; Scott, Joseph W. ; Bauzo, Rayna M.; Xiang, Zhimin; Richards, Nigel G.; Haskell-Luevano, Carri e. (2004) Epimeriza tion Studies of the Monocyclic Agouti-Related Protein(103-122) Arg-Phe-Phe Residues: Conversion of the Melanocortin-4 Receptor Antagonist into an Agonist and Results in the Discovery of a Potent and Se lective Melanocortin-1 Agonist. Journal of Medicinal Chemistry in Press 201 Schioth, H.B., Muceniece, R., Wikber g, J.E.S. and Chhajlani, V. (1995) Characterization of Melanocortin Recep tor Subtypes by Radioligand Binding Analysis. European Journal of Pharmacology -Molecular Pharmacology Section 288 (3), 311-317 202 Qu, S.Y., Yang, Y.K., Li, J.Y., Zeng, Q. and Gantz, L. (2001) Agouti-related protein is a mediator of diabetic hyperphagia. Regulatory Peptides 98 (1-2), 69-75

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186 BIOGRAPHICAL SKETCH Christine G. Joseph is from a beautiful Caribbean place called Carriacou, a 13square-mile island belonging to Grenada. Sh e was born there, to Durrant I. Joseph and Grace E. Joseph. She graduated from Dover primary school and BishopÂ’s College (a secondary school on the island) before leavi ng for the US. Shortly after arriving in the US, Christine enrolled at SUNY-New Pa ltz in New Paltz, New York (fall 1993). Christine received her BS in chemistry with a minor in mathematics in 1997, and graduated with honors. Not sure if she was r eady for graduate schoo l after graduation, she worked for 2 years in the pharmaceutical indus try before joining the Medicinal Chemistry department at the University of Florida in August 2000. There she pursued her doctoral studies under the supervision of Dr. Carrie Haskell-Luevano. Chri stineÂ’s future plans include relocation so that she can advan ce her scientific career in a postdoctoral appointment.