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Structure-Activity Relationships of an Agouti-Related Protein-Derived Decapeptide at the Murine Melanocortin Receptors


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STRUCTURE-ACTIVITY RELATIONSHIPS OF AN AGOUTI-RELATED PROTEINDERIVED DECAPEPTIDE AT THE MURINE MELANOCORTIN RECEPTORS By ANZEELA MULAIYA SCHENTRUP A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Anzeela Mulaiya Schentrup

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This document is dedicated to my hus band, Joseph, not only for being a loving, supportive and dedicated partner, but for being mom and dad to our kids and to me when I needed it. Any success I have is because of you.

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ACKNOWLEDGMENTS I owe thanks to many people for their role in the completion of this work. In particular, I acknowledge Carrie Haskell-Luevano, PhD, my mentor for this project, for her guidance and willingness to support me in my career choices. I hope always to have a professional relationship with her, and I admire her accomplishments and dedication to her work and students. Also, I thank Julie Johnson, PharmD, for encouraging me to see this project through and for showing me my place in the field of pharmacy sciences. I cannot say enough about how important her professional influence has been. I recognize Margaret James, PhD, for allowing me this opportunity to complete this project. And finally, my thanks go to Kenneth Sloan, PhD, for directing me into the exciting area of pharmacy research in the first place and helping me with some of the most important career decisions that I have made. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .......................................................................................................................ix CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 Focus on the Melanocortin System Components.........................................................2 Solid Phase Peptide Synthesis: "FMOC" Chemistry....................................................6 2 MATERIALS AND METHODS.................................................................................9 Synthesis of the Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr Cyclic Decapeptide..............................................................................................................9 Solid Phase Peptide Synthesis Preparation............................................................9 Manual Solid Phase Peptide Synthesis................................................................11 Removal of Orthogonal Protecting Groups and Cleavage of the Peptide from the Resin...........................................................................................................13 Analysis of the Linear Peptide............................................................................14 Cyclization of the Linear Peptide........................................................................15 Analysis of the Cyclized Peptide.........................................................................15 Biological Assays.......................................................................................................15 Cell Culture and Transfection.............................................................................15 Receptor Binding Assays....................................................................................16 Quantification of the Receptor Binding Assays..................................................16 -galactosidase Bioassay.....................................................................................17 Quantification of -galactosidase Bioassay.........................................................17 Analysis of Bioassay Data...................................................................................18 3 RESULTS...................................................................................................................19 Receptor Binding........................................................................................................19 Receptor Antagonist Activity.....................................................................................19 v

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Receptor Agonist Activity..........................................................................................20 4 DISCUSSION AND CONCLUSION........................................................................21 Discussion...................................................................................................................21 Conclusion..................................................................................................................24 CHEMICAL STRUCTURES............................................................................................25 REFERENCES..................................................................................................................28 BIOGRAPHICAL SKETCH.............................................................................................32 vi

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LIST OF TABLES Table page 1-1 Melanocyte-stimulating hormone peptide structures...................................................2 2-1 Amino acids used and quantities..................................................................................9 2-2 Protocol for manual FMOC chemistry synthetic strategy..........................................11 A-1 Chemical structures for FMOC amino acids..............................................................25 A-2 Chemical structures for FMOC reagents....................................................................27 A-3 Various reagents for Solid Phase Synthesis................................................................27 vii

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LIST OF FIGURES Figure page 1-1 Alignment of Human ASP and Human AGRP in ClustalW 24 The C-terminus is in bold-face and the portion used to derive the decapeptide is indicated in italics. The common Arg-Phe-Phe (RFF) motif is shaded.....................................................5 2-1 This glass fritted filter glass container contains a stem with a bidirectional valve. The filter flask is attached to a ring stand and placed above and connected to a waste flask. The bidirectional valve is connected to a nitrogen gas line and the waste flask is attached to a vacuum source..............................................................10 2-2 An example of the deprotection scheme using FMOC chemistry. R = resin bead....12 2-3 Activation of the coupling reaction by BOP...............................................................13 2-4 Cleavage of the peptide chain from the resin bead by TFA........................................14 3-1 -galactosidase expression is given for varying concentrations of decapeptide with the varying concentrations of MTII.........................................................................20 3-2 -galactosidase expression is given for varying concentrations of decapeptide peptide concentration. For comparison, the -galactosidase expression for MTII is given.....................................................................................................................20 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science STRUCTURE-ACTIVITY RELATIONSHIP OF AN AGOUTI-RELATED PROTEIN-DERIVED DECAPEPTIDE AT THE MURINE MELANOCORTIN RECEPTORS By Anzeela Mulaiya Schentrup December 2005 Chair: Carrie Haskell-Luevano Major Department: Medicinal Chemistry Obesity is a complex, multi-factorial chronic disease involving several contributing factors. The melanocortin neurohormone messenger system is thought to play a role in modulating the obesity phenotype. In particular, Agouti-related protein (AGRP), an endogenous antagonist of melanocortin receptors, interacts with the human brain melanocortin receptors hMC4R and hMC3R. When AGRP is over-expressed centrally in transgenic mice, the result is an obese phenotype. Thus, AGRP expression in the hypothalamus is thought to be involved in the regulation of energy homeostasis at the brain melanocortin receptors. We hypothesized that the AGRP antagonist activity is determined by the structure-function relationship between specific amino acids in AGRP and its receptor. The decapeptide, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr, which is derived from the AGRP C-terminal domain, has been reported to possess M antagonistic properties at the hMC4R. We synthesized this decapeptide using a manual solid-phase ix

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peptide synthetic strategy. In addition, we pharmacologically characterized this decapeptide at the murine melanocortin receptors, mMC1R, mMC3R, mMC4R and mMC5R. In particular, we used the competitive displacement of I 125 -MTII, a radio-labeled melanocortin agonist, to determine binding and the -galactosidase bioassay to determine receptor activity. We found the decapeptide, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr, to be a weak antagonist at the mMC4R (pA 2 = 6.79) and to possess agonist activity (2.89 2.26 M) at the skin mMC1R. Further, the decapeptide did not have appreciable activity at the mMC3R or mMC5R. The agonist activity at the mMC1R skin receptor was unexpected and contrasts with a previous report that AGRP has no activity at the MC1R. Our study provides experimental evidence that the C-terminal portion of AGRP contains a specific recognition sequence for melanocortin receptor activity. Furthermore, we conclude that the conformational shape which the AGRP protein attains is a significant determinant of AGRP activity at the melanocortin receptors. x

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CHAPTER 1 BACKGROUND AND SIGNIFICANCE Obesity is a highly prevalent chronic disease in humans. Based on data from the National Health and Nutrition Examination Survey (NHANES) from 1999-2002, 30 percent of adults 20 years of age and older (greater than 60 million people), had a body mass index (BMI) of 30 or greater. This is significantly increased from 23 percent in 1994. 1 The high prevalence of obesity gives rise to increasing numbers of patients with obesity-related complications such as musculoskeletal disorders, cardiovascular disease, decreased respiratory function and diabetes mellitus. These sequelae have a significant negative impact on patients quality of life as well as on health care cost. Furthermore, because of these serious complications, obesity is now considered the second leading cause of preventable death in the U.S. 1, 2 Current research on controlling obesity in humans involves focus on the factors involved in obesity development. Several factors, including genetic make-up, physiology, environment, behavior and individual psychology all modulate obesity risk and severity. In particular, previous studies identify the function of neuroendocrine messenger systems such as the melanocortin system as a genetic and physiological factor which plays an important role in human weight homeostasis in both animal models and humans 3-6 Specifically, the melanocortin messengers interact with several other central and peripheral neurohormone messengers to affect weight homeostasis by modifying feeding behavior and thermogenesis. By characterizing the key structural elements of the components of the melanocortin system, researchers hope to uncover a powerful therapeutic target for drug 1

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2 therapy in an effort to control obesity in humans. The following is a brief overview of the melanocortin system and how it is purported to fit into the complex system of central weight regulation. Focus on the Melanocortin System Components The melanocortin system consists of melanotrophic peptides, endogenous peptide inhibitors and receptors. In particular, the melanotrophic peptide hormones are derived from the proopiomelanocortin (POMC) gene transcript. POMC is the source of multiple hormones including adrenocorticotropin (ACTH), -lipotropin, met-enkephalin and -endorphin 7 The POMC product that is produced at a given time depends on the hormone system that activates POMC transcription. When POMC is activated and transcribed as part of melanocortin activation, it gives rise to -, and melanocyte stimulating hormone (Table 1-1). Most research has focused on -melanocyte stimulating hormone Table 1-1 Melanocyte-stimulating hormone peptide structures Chemical Sequences -MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH 2 -MSH NH 2 -Asp-Glu-Gly-Pro-Tyr-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-OH -MSH NH 2 -Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH (-MSH), a 13-amino acid peptide (Figure 1-1) which is the prototype endogenous agonist of the melanocortin system receptors. It is produced both centrally and peripherally and has been found to stimulate the five melanocortin receptor subtypes. The melanocortin receptors are G-protein coupled receptors (GPCRs). They have the classic GPCR secondary structure consisting of seven lipophilic -helical membrane-spanning regions. The N-terminus is exposed on the extracellular portion of the membrane while the C-terminus remains intracellular. In this construct, the melanocortin peptides putatively interact with the helical pocket that is formed by the surrounding

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3 transmembrane regions of the receptor. Interaction of the ligand and receptor induces a conformational change in the target receptor resulting in activation of the receptor. Activated melanocortin receptors interact with a trimeric G-protein. In the case of the melanocortin receptors, this interaction stimulates G s which activates adenylate cylase (AC). AC triggers an intracellular cascade of signaling in which adenosine triphosphate (ATP) is converted to cyclic adenosine monophosphate (cAMP). Cyclic AMP then acts as a second messenger and modulates subsequent physiological activity 7 Specifically, melanocortin receptor activation causes an increase in cAMP-mediated phosphokinase A (PKA) activity which activates the cAMP response element binding protein (CREB). Then, CREB interacts with cAMP response element (CRE) on deoxyribonucleic acid (DNA) to activate cell-specific transcription of mRNA resulting in a physiological response. Five subtypes of melanocortin receptors in several species have been identified and are numbered in the order in which they were originally cloned. Further, murine receptor isolates are homologous to human receptors 8 The melanocortin 1 receptor (MC1R) is located peripherally in both humans and mice and is found in melanocytes, melanoma cells and macrophages. MC1R is known to affect pigmentation and are reputed to be involved in the inflammatory response 9 however all of the mediators and specific effects of MC1R involvement are as yet unknown. The melanocortin 2 receptor (MC2R) is responsive only to ACTH and is located in all three layers of adrenal cortex as well as on adipocytes. These receptors are involved in adrenal steroidigenesis and also have adrenocorticotrophic effects 10 The melanocortin 3 receptor (MC3R) is expressed in various areas, particularly in the brain, including the arcuate nucleus and the anterior

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4 ventral periventricular nucleus. In addition, the MC3R is expressed in the heart, placenta, pancreas and gut. MC3R, like the melanocortin 4 receptor (MC4R), discussed below, plays a role in weight homeostasis 11 The melanocortin 5 receptor (MC5R) is expressed ubiquitously in muscle, liver, spleen, lung, brain, adipocytes, and a variety of other tissues. Deletion of MC5R in animal experiments results in exocrine gland dysfunction, however more research regarding its specific function is needed 12 The MC4R is primarily expressed in the CNS, specifically in cortex, hippocampus, thalamus and hypothalamus 13 In terms of function, MC4R is involved in feeding behavior and weight homeostasis in both animal and human models. MC4R knockout mice develop obesity, hyperphagia, hyperinsulinemia and hyperglycemia, indicating that receptor activation is important in inducing satiety and preventing obesity 14 The melanocortin system is currently the only GPCR system that is known to involve both endogenous receptor agonists and antagonists. In particular, the agouti gene locus (ASIP) encodes Agouti Signaling Protein (ASP), a known antagonist of the melanocortin receptors in animal models 15 This protein is expressed in the periphery and is responsible for the phenotype of the lethal A y mouse, in which ASP is ectopically expressed. Phenotypically, lethal A y mice are obese with a yellow coat color 16 as a result of this ectopic ASP expression. The yellow coat color is a result of a shift in production of eumelanin to pheomelanin 17 that occurs as a result of ASP interaction with MC1R. Also, ASP antagonizes central melanocortin receptors in lethal A y mice, leading to the obesity trait. Although this antagonism occurs centrally, ASP is not known to be expressed endogenously in the central nervous system. 18 Researchers have used this

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5 result to look for a protein analogous to ASP that is produced centrally and may contribute to the obese phenotype in mice and humans. Homology studies with ASP in several species lead to the discovery of Agouti-Related Protein, or AGRP, which is a centrally-expressed analog to ASP in both murine and human models 19 ASP and AGRP are 131-amino-acid proteins in mouse and 132-amino-acid proteins in humans. Further, both have cysteine-rich C-termini with secondary structure that is maintained through disulfide bridges 19-21 The sequence homology between ASP and AGRP is 40% percent in the C-terminus 19 (Figure 1). AGRP is known to bind and antagonize both MC3R and MC4R, while ASP is active at both MC1R and MC4R 15, 22, 23 Previous research has identified the triplet of amino acids Human ASP MDVTRLLLATLLVFLCFFTANSHLPPEEKLRDDRSLRSNSSVNLLDVPSV 50 Human AGRP -----MLTAAVLSCALLLALPATRGAQMGLAPMEGIRRPDQALLPELPGL 45 SIVALNKKSKQIGRKAAEKKRSSKKEASMKKVVRPRTPLSAP-CVATRNS 99 GLRAPLKKTT--AEQAEEDLLQEAQALAEVLDLQDREPRSSRRCVRLHES 93 CKPPAPACCDPCASCQCRFFRSACSCRVLSLNC-----132 CLGQQVPCCDPCATCYCRFFNAFCYCRKLGTAMNPCSRT 132 Figure 1-1 Alignment of Human ASP and Human AGRP in ClustalW 24 The C-terminus is in bold-face and the portion used to derive the decapeptide is indicated in italics. The common Arg-Phe-Phe (RFF) motif is shaded. at positions hAGRP (111-114), Arg-Phe-Phe, as necessary for MC4R antagonism. 25 Further, structural studies indicate that these residues form an active loop region in the peptide which interacts with the melanocortin receptors 20 Short cyclic fragments of the C-terminus of ASP and AGRP that contain the Arg-Phe-Phe domain were shown by Tota et al to possess potent antagonist activity at MC4R but 10-fold lower activity and binding than that of the full C-terminus 25 In addition, no activity was seen for these fragments at MC3R.

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6 The purpose of the current study is to identify binding interactions between synthetic melanocortin receptor antagonists and the melanocortin receptors in order to logically design molecules to modulate the activity of melanocortin receptors and also to provide information about the structure of these receptors and antagonists in vivo. Specifically, this study looks at the interaction of a synthetic cyclic decapeptide derived from AGRP with all five murine melanocortin receptors. A brief overview of the synthetic strategy used to synthesize the test compound follows. The specific synthetic scheme is given in the Methods section of Chapter 2. Solid Phase Peptide Synthesis: "FMOC" Chemistry Solid-phase peptide synthesis was conceived by R.B. Merrifield in 1959 26 The strategy involves the covalent attachment of a nascent peptide chain to an insoluble polymeric support resin. The resin is maintained in a filter system that allows the peptide to be anchored and easily separated from reaction reagents. This procedure allows the use of excess soluble reagents which can be filtered and washed from the reaction vessel once amino-acid coupling reactions have terminated. The use of excess reagents allows the reactions to be driven to high yields (>90%). In addition, the catalysis of these reactions minimizes the formation of side products. By using a series of deprotection and coupling steps, the reasearcher can select specific reactive moieties on the peptide to participate in chemical reactions while others remain protected. Since this rationally designed system of synthesis is generous in allowing high concentrations of reagents and the synthesized molecule is easily separated from reagents, the system lends itself well to automation 27 Synthetic methods of solid phase peptide synthesis are differentiated based on the protection scheme used. Protection schemes require the use of both N-and side chain

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7 protection moieties so that the exposed reactive portions of the nascent peptide can be manipulated separate from each other. During peptide synthesis, temporary N-protecting groups are removed with each amino-acid coupling step to reveal the portion of the amino acid which will be coupled to the next residue. Permanent side chain protecting groups are used to protect vulnerable side chain groups during coupling reactions and are removed altogether after synthesis has been completed. The FMOC synthetic strategy was used to synthesize the test compounds in this study. This strategy is based on an orthogonal system of protecting groups developed by Carpino and Han 28 Orthogonal schemes are based on using two or more classes of protecting groups that can be removed by differing chemical mechanisms. FMOC chemistry utilizes molecules constructed using a base labile temporary N-9-fluorenylmethyloxycarbonyl (FMOC) group. The permanent protecting groups are primarily based on tertiary-butanol esters and ethers which provide bulky, base-stable protection to otherwise labile amino acid side chains. During synthesis, the temporary FMOC group is removed sequentially after each amino acid is added in order to form the growing peptide chain while the permanent protecting groups remain intact until the end of synthesis 29 The FMOC synthetic scheme described above is used in this study to produce synthetic peptides for use in research on the melanocortin system. In particular, this study is focused on Agouti-related protein (AGRP), a component of the melanocortin system that is of particular interest as an obesity target. In particular, previous research establishes that AGRP participates in normal signaling for human feeding behavior and energy homeostasis 30 Therefore, structural alterations in AGRP may affect this normal

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8 signaling and thereby contribute to the development of obesity. To structurally characterize the activity of AGRP at the melanocortin receptors, biochemists have isolated the cyclic decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr from the AGRP C-terminus 25 According to Tota et al., this small portion of the AGRP molecule binds to and antagonizes the human MC4R. In order to verify this finding which establishes the cyclic decapeptide as the active portion of AGRP and further analyze its interaction with melanocortins receptors that were previously unstudied, we synthesized the decapeptide using manual solid phase peptide synthesis using the FMOC strategy described by Stewart et al. 31 We then pharmacologically characterized the peptide at the 5 murine melanocortin receptors. We used the murine model of melanocortin receptor action to test our hypothesis that the cyclic decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr is the minimally active fragment of the AGRP molecule and will thus have antagonist properties at the mMC3R and mMC4R. Further, the decapeptide will not have significant activity, similar to its parent AGRP, at other murine melanocortin receptors.

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CHAPTER 2 MATERIALS AND METHODS The synthesis of the cyclic decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr was accomplished by manual solid phase peptide synthesis using the FMOC synthetic strategy and then tested in vitro for binding and activity at the murine melanocortin receptors. Synthesis of the Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr Cyclic Decapeptide Solid Phase Peptide Synthesis Preparation Reaction reagents and their structures are listed in the Appendix and corresponding reaction quantities are shown in Table 2-1. All peptide reagents and resin were obtained from Peptides International (Louisville, KY, USA). All reagents and solvents were American Chemical Society (ACS) grade or higher. Table 2-1 Amino acids used and quantities Amino Acid Protection Scheme Equivalents Molar quantity (mmol) Molecular Weight Amount (g) Cys FMOC/Trt 3.13 0.76 585.73 0.45 Phe FMOC 3.13 0.76 387.44 0.29 Ala FMOCH 2 O 3.13 0.76 329.36 0.25 Asn FMOC/Trt 3.13 0.76 596.69 0.45 Arg FMOC/Pbf 3.13 0.76 648.78 0.49 Tyr FMOC/tBu 3.13 0.76 459.55 0.35 The manual FMOC synthesis was conducted in a reaction vessel consisting of a glass-fritted filter and a valve separating two ports. First, the glass reaction vessel was acid-washed with chromerge and concentrated H 2 SO 4 The vessel was then silanized using 10% dichloromethylsilane (DCMS) in dry toluene and mixed using helium gas for 9

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10 15 to 30 minutes. After mixing, the reaction vessel was rinsed with toluene, then treated with dry methanol for 15 minutes, and then dried with acetone. Upon assembly of the reaction apparatus, one port of the reaction vessel was attached to nitrogen gas and the other to a vacuum line for waste removal. The reaction vessel was then mounted to a ring stand inside a vertical laminar flow hood. A waste flask was secured beneath the reaction vessel (Figure 2-1). The gas and vacuum lines were primed. Figure 2-1 This glass fritted filter glass container contains a stem with a bidirectional valve. The filter flask is attached to a ring stand and placed above and connected to a waste flask. The bidirectional valve is connected to a nitrogen gas line and the waste flask is attached to a vacuum source. FMOC-Wang resin 32 beads (Appendix) were added to the pre-weighed reaction vessel and weighed. Since the dry polystyrene resin beads have an average diameter of 50 m, the beads were then soaked in dimethylformamide (DMF) for two hours to promote swelling. After this process, the beads obtained 2.5 to 6.2 times their dry volume and formed a well-solvated gel. This maximally increases the bead surface area and exposes the reagent-accessible chemical attachment points.

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11 Manual Solid Phase Peptide Synthesis The synthesis procedure is outlined in Table 2-2. After copious rinsing of the Table 2-2 Protocol for manual FMOC chemistry synthetic strategy Cycle Function Time 1 DMF wash 5 X 1min 2 20% Piperidine in DMF rinse 2 min 3 20% Piperidine in DMF deprotection 18 min 4 DMF wash 4 X 1 min 5 Ninhydrin Test 3 min 6a-d Add : FMOC-AA dissolved in DMF (3 eq.) BOP (3 equiv.) HOBt (3 equiv.) DIEA (5.1 equiv.) 7 Coupling 8 Ninhydrin Test 3 min 9 DMF wash 3 X 1 solvated resin with DMF, the beads were rinsed with 20% piperidine for 2 minutes, drained, and then mixed again for 18 minutes in 20% piperidine to remove the FMOC protecting group (Figure 2-2). The resin was then rinsed with DMF and the Kaiser ninhydrin test 33 (described below) was conducted to observe the extent of deprotection. For the ninhydrin test, a small sample of the resin was removed by capillary pipette and placed into a small glass vial. Two drops of each of the Kaiser ninhydrin test solutions A, B and C (Appendix) were added to the vial. The vial was then heated to approximately 100C for 3 minutes. A positive test indicating complete deprotection of the free peptide chain amino group was given by a homogeneous deep blue color. If a negative test was obtained, the piperidine deproctection step was repeated until the ninhydrin test was positive.

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12 N -9-florenylmethyloxycarbonyl protecting group (FMOC) NH Glycine Aspartic Acid Tyrosine (Gly)(Asp) (Tyr)Piperidine RONHOOONHONHOOHOOCH3CH3CH3CH3CH3CH3O Fmoc-Tyr(tBu)-Wang resin ONHOOONHONH2OOCH3CH3CH3CH3CH3CH3O R Fmoc-Tyr(tBu)-Wang resinCOO Carbon Dioxide GasFree Amino GroupCH2 N+HH Figure 2-2 An example of the deprotection scheme using FMOC chemistry. R = resin bead. After confirmation of successful deprotection, the next amino acid in the decapeptide sequence was added to the reaction vessel in the amount given in Table 2.1. In addition, benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) in 3-fold excess (Appendix) was added to accelerate the formation of the -Obt ester form of the FMOC amino acids (Figure 2-3). Also, 1-hydroxybenzotriazole (HOBt) (Appendix) in 3-fold excess was used for three reasons: (1) to accelerate the cabodiimide-mediated couplings, (2) to suppress racemization and (3) to inhibit dehydration of the carboxamide side chains of Asn and Gln to the corresponding nitriles. Finally, N,N-diisopropylethylamine (DIEA) in 3.1 fold excess was used to stabilize side products of the amino acid coupling reaction (Figure 2.3). Completion of the amino acid

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13 N(CH ) O 3 2 H O N Figure 2-3 Activation of the coupling reaction by BOP. addition and FMOC deprotection was monitored using the ninhydrin test. After repeating the procedure nine times to complete the linear peptide Tyr-Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys-Tyr, the reaction vessel containing the resin and newly formed peptides was dried under vacuum overnight. The weight of the peptides and resin combination was determined after drying was complete. Removal of Orthogonal Protecting Groups and Cleavage of the Peptide from the Resin The orthogonal protecting groups on the peptide amino acids were cleaved from the peptide in the same step as the peptide cleavage from the resin beads. This was accomplished using a trifluoroacetic acid (TFA), 1,2 ethanedithiol, p-cresol, and water cleavage cocktail in a 15:3:1:1 ratio (Figure 2-4). N N N O P + N(CH 3 ) 2 (H 3 C) 2 N (H 3 C) 2 N P F F F F F F NH + C3 H C H 3 C3 H C H 3 C3 H N H O O R 1 P + N(CH 3 ) 2 N(CH 3 ) 2 (H 3 C) 2 N FMOC N H O O R 1 FMOC HOBt -OBt OBt R 1 FMOC P N(CH 3 ) 2 (H 3 C) 2 N BOP benzotriazol-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate N N2 H R N H O R 1 FMOC N H R DIE A N H O R 1 FMOC O N H O R 1 FMOC O P N(CH 3 ) 2 N(CH 3 ) 2 (H 3 C) 2 N N N -OBt DIEA Diisopropylethylamine Fmoc Amino Aci d Tertiary Amine Base N N N O -OBt O H Growing Deprotected Peptide DIE A Coupled Peptide Symmetric Anhydride HOBt-active ester hexamethylphosphoramide

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14 Linker + Resin Figure 2-4 Cleavage of the peptide chain from the resin bead by TFA. Five milliliters of the cleavage cocktail were added to a medium round bottom flask with the dried resin and allowed to mix for 2 hours at 0C and then 30 to 45 minutes at room temperature. The decapeptide was then separated from the resin by filtration and the resulting solution was concentrated. After concentration, the peptide was extracted using water and ethyl ether and the extract was concentrated until a white flocculent appeared. The resulting crude peptide was then lyophilized. Analysis of the Linear Peptide The success of the manual linear peptide synthesis was confirmed using high performance liquid chromatography (HPLC) purification with a Shimadzu chromatography system with a photodiode array. The structure was confirmed with gas chromatography/ mass spectroscopy (GC/MS). Glycine (Gly) Tyrosine (Tyr) O F F F O H Trifluoroacetic Acid (TFA) O O Cysteine (Cys) O O N H O O N H N H OH O C3 H C H 3 C H 3 S C H 3 C H 3 C3 H R

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15 Cyclization of the Linear Peptide Upon confirmation of the weight of the linear peptide by analytical methods, a disulfide bridge was formed 34 between the two cysteine residues on the linear decapeptide Tyr-Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys-Tyr. Half of the crude peptide was dissolved in 1.5 L 0.1% degassed acetic acid in water. The pH was adjusted from 3.3 to 8.5 with concentrated ammonium hydroxide and the solution was then mixed overnight. After mixing, the pH of the solution was lowered to 4.0 with glacial acetic acid. A non-measured amount of HCl-amber-lite ion exchange resin was added and this combination was mixed until the yellow solution turned clear (~1 hour). The exchange resin was then filtered into a large round bottom flask and concentrated. The resulting compound, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr was lyophilized. Analysis of the Cyclized Peptide The final cyclized product was analyzed by MALDI-TOF mass spectroscopy at the University of Florida Protein Core Facility. Final peptide purification was achieved using a semi-preparative RP-HPLC C 18 bonded silica column (Vydac 218TP1010, 1.0 X 25 cm). The structure was assessed by analytical RP-HPLC and 2D 1 H NMR. (University of Florida protein core facility). Biological Assays Cell Culture and Transfection HEK-293 cells (human embryo kidney cells transformed with human adenovirus type 5) were maintained in Dulbeccos modified Eagles medium (DMEM), 10% penicillin-streptomycin and 10% fetal calf-serum. The cells were seeded 1 day prior to transfection at 1-2 x 10 6 cells/100mm. Murine melanocortin receptor cDNA (coding deoxyribonucleic acid) (20 g) cloned into in the pcDNA3 expression vector in a

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16 previous experiment was transfected into the seeded HEK-293 cells using the calcium phosphate method 35 G418 sulfate aminoglycoside (C 20 H 40 N 4 O 10 .2H 2 SO 4 ) selection 36 was used to isolate stable receptor populations (1 g/mL). Receptor Binding Assays HEK-293 cells that were transfected and were stably expressing the various melanocortin murine receptors (mMC1R, mMC3R, mMC4R and mMC5R) were maintained in Dulbeccos modified Eagles medium (DMEM), 10% penicillin-streptomycin and 10% fetal calf-serum. One day prior to conducting the binding studies, transfected cells were plated into Primera 24 well plates (Falcon) at a density of 0.1-0.3 x 106 cells perwell. Concentrations of 10 -6 to 10 -12 M MTII (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH 2 ), a potent melanocortin receptor agonist 37 and 10 -4 to 10 -10 M Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr were used to competitively displace MTII that was radio-labeled with I 125 (100000 cpm/well; NEN Life Sciences). A 450 L solution of the concentration of decapeptide being tested was added to each well. Next, 50 L of I 125 MTII was added to each well and incubated at 37C for 1 hour. The medium was then rinsed and washed with assay buffer (DMEM, 0.1 mg/ml and Bovine Serum Albumin (BSA)). The cells were lysed with 0.5 mL 0.1 M NaOH and 0.5 mL 1% Triton X-100 for 10 minutes, and then transferred to 16 X 150 mm glass tubes. Quantification of the Receptor Binding Assays The binding of I 125radiolabeled MTII was measured using a -counter. Dose-response curves and IC 50 values for 10 -6 to 10 -12 M MTII and 10 -4 to 10 -10 M Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr were generated and analyzed by nonlinear least-squares analysis. The IC 50 values represent the mean ( + standard deviation) of duplicate wells generated in at least two independent experiments.

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17 -galactosidase Bioassay HEK-293 cells stably expressing melanocortin receptors (mMC1R, mMC3R, mMC4R, mMC5R) were transfected with 4 g of the CRE/-galactosidase reporter gene 38 using the same transfection method described above. Primera 96-well plates were treated with 5000-15000 post-transfection cells and incubated overnight. Forty-eight hours post-transfection, cells were stimulated with MTII (with 10 -6 to 10 -12 M), Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr (10 -4 to 10 -10 M) and forskolin (10 -4 M) in assay medium (DMEM containing 0.1mg/mL BSA and 0.1 mM isobutylmethylxanthine) for 6 hours. The assay medium was aspirated and 50 L of lysis buffer (250 mM TrisHCl, pH 8.0, and 0.1% Triton X-100) was added to each well. The plates were then stored at -80C overnight. The next day, the plates containing the cells lysates were thawed and10 L aliquots were taken from each well and transferred to another 96-well plate. The 10 L samples were set aside for relative protein determination. Phosphate-buffered saline (PBS) with 0.5% BSA (40 L) was then added to each well of the original plates of cell lysate. Further, 150 L of substrate buffer (60 mM sodium phosphate, 1 mM MgCl 2 10 mM KCl, 5 mM -mercaptoethanol, 200 mg/mL 2-Nitrophenyl--D-galactopyranoside (ONPG)) was added to each well and the plates were incubated at 37C. Quantification of -galactosidase Bioassay The sample absorbance OD 405 was measured using a 96-well plate reader. The relative protein value was determined by adding 200 L 1:5 dilution of G250 protein dye (Bio-Rad):water to the 10 L cell lysate aliquots taken previously. In addition, the OD 595 was measured. Furthermore, the transfection efficiency of the CRE--galactosidase reporter assay was determined using 10 -4 M forskolin treatments of 6 wells of each plate as controls. Data points were normalized both to the relative protein content and non-.

PAGE 28

18 Analysis of Bioassay Data Data analysis was conducted and IC 50 and EC 50 values were determined by using nonlinear regression analysis with the PRISM program (v2.0, GraphPad Inc.). In addition, pA 2 values quantifying antagonism were generated using the Schild analysis method. 39 The EC 50 and pA 2 values represent the mean (+standard deviation) of triplicate wells examined in at least two independent experiments.

PAGE 29

CHAPTER 3 RESULTS The synthesis of the melanocortin decapeptide, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr, was conducted with a yield of 0.5g of product. We confirmed that each of the coupling steps in the synthesis was completed using the ninhydrin test as previously described. The purified peptide was > 99% pure and the sequence was determined by methods described previously to be the sequence of the desired decapeptide with the correct molecular weight. Both the binding activity and function of the decapeptide at the murine melanocortin receptors was determined. Receptor Binding The hAGRP decapeptide bound extensively to the mMC4R (IC 50 = 275 62 nM). Additionally, the decapeptide possessed M binding IC 50 values at mMC3R (IC 50 = 11.7 3.9 M), mMC5R (IC 50 = 38.7 23.5 M), and mMC1R (IC 50 = 3.09 2.19 M). Receptor Antagonist Activity The hAGRP decapeptide was an antagonist of mMC4R (pA2 = 6.8 0.4) (Figure 3-1). In addition, it has only slight antagonist activity at the mMC3R which was not significant enough to quantify, and lacked antagonist activity at mMC5R at concentrations up to 100 M. 19

PAGE 30

20 Receptor Agonist Activity The hAGRP decapeptide was found to have agonist activity at mMC1R (EC 50 = 2.89 2.26 M) (Figure 3-2).. No agonist activity was detected for the decapeptide at any of the other melanocortin receptors (mMC3R, mMC4R, mMC5R). Figure 3-1 -galactosidase expression is given for varying concentrations of decapeptide with the varying concentrations of MTII. Figure 3-2 -galactosidase expression is given for varying concentrations of decapeptide peptide concentration. For comparison, the -galactosidase expression for MTII is given.

PAGE 31

CHAPTER 4 DISCUSSION AND CONCLUSION Discussion Our most significant finding in this study is that the AGRP decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr has both unexpected agonist activity at the mMC1R while also being an antagonist of the mMC4R. The antagonist activity at mMC4R that we observed is consistent with our hypothesis and previous research 30 however, the agonist activity at mMC1R has not been described in previous studies. Furthermore, our findings imply that the conformational space that AGRP occupies in vivo is related to the activity of AGRP at the melanocortin receptors. The finding that the AGRP decapeptide has activity at the mMC1R is in contrast with previous research which has shown that the parent AGRP protein has no activity at the skin mMC1R 22 These previous studies are supported by the finding that transgenic mice ectopically expressing AGRP have the brown (wild-type) coat phenotype. This previous result indicates that AGRP does not shift the production of eumelanin to pheomelanin in these transgenic mice and, thus, AGRP has no activity at skin mMC1R. In our study, we also expected to find that the AGRP decapeptide that we studied would have no agonist activity at the mMC1R. However we observed that the AGRP decapeptide has micromolar agonist activity at the mMC1R and sufficient binding at this receptor (3.09 2.19 M) to explain this activity as a direct receptor effect. Since the AGRP decapeptide amino acid sequence is derived from the sequence of its parent protein, AGRP, the contrasting activity between the decapeptide and its parent 21

PAGE 32

22 peptide at the mMC1R cannot be explained by the decapeptide amino acid sequence alone. Specifically, the decapeptide possesses the identical amino acid sequence as the active portion of the human and murine AGRP C-terminus (108-177) 25 Although the decapeptide has some distinct chemical moieties in analogous positions of the human and murine Agouti Signal Protein (Figure 4.1), the decapeptide retains the Arg-Phe-Phe motif in a conserved position between AGRP and ASP. This Arg-Phe-Phe sequence is thought to be essential for the antagonist activity of the melanocortin antagonists 40 Moreover, in contrast to AGRP, when mASP is ectopically expressed in transgenic mice, mice with the agouti coat color are produced. The resulting agouti coat color indicates that mASP is an antagonist at the skin mMC1R 41 Thus, although the AGRP decapeptide sequence is derived from a consensus sequence of the AGRP and ASP C-terminus, the AGRP decapeptide itself has different pharmacological activity than both of its parent proteins. Previous research has indicated that the conserved Arg-Phe-Phe (RFF) motif found in both the ASP and AGRP active sites may be important for receptor recognition of these endogenous antagonists. In support of this idea, studies have shown that mutation of these Arg-Phe-Phe residues in AGRP results in considerably less efficacious antagonism at the melanocortin receptors. 4 Furthermore, homology exists between the Arg-Phe-Phe motif found in the melanocortin endogenous antagonists and the conserved His-Phe-Arg-Trp residues in the endogenous melanocortin ligands. Specifically, the Arg-Phe-Phe motif may be mimicking the His-Phe-Arg-Trp molecular interactions and allowing the melanocortin receptors to recognize AGRP and ASP. Moreover, previous studies have found that the tetrapeptide AcHis-Phe-Arg-Trp -NH 2 is the minimal fragment of melanocortin agonists required to produce a physiological response (M) in

PAGE 33

23 the classic frog skin bioassay 42 Therefore, our study provides the first experimental data to support the hypothesis that the conserved antagonist Arg-Phe-Phe residues may be mimicking the agonist His-Phe-Arg-Trp interactions with melanocortin receptors, specifically at the skin mMC1R. Our finding that the decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr has agonist activity at MC1R is consistent with a model in which the conformational shape that AGRP adopts determines its activity at its receptor. Since the decapeptide was synthesized to mimic a single portion of the AGRP C-terminus, our study supports the hypothesis that the amino acids adjacent to the decapeptide residues on the parent AGRP peptide influence the conformational shape of the entire peptide. Thus, the spatial relationships between the antagonist residues and the residues on the receptor are important for determining the activity of AGRP at the melanocortin receptors. Some experimental characteristics of our study resulted in some differences in our findings and those from previous research in human melanocortin receptors and may limit the comparison between our results and those previous. In particular, the binding affinity that we found for the hAGRP at the mMC3R is 10-fold less potent than the binding affinity previously reported for hMC3R. In addition, the binding affinity we report for mMC4R is 4-fold less potent than the binding affinity reported for hMC4R. We attribute these differences to two main experimental differences between our study and previous studies: the species of receptor used and the differences in the radio-labeled compound used. In our research, we used murine melanocortin receptors with the intention of further reproducing our results in vivo in murine models, whereas previous researchers have used human melanocortin receptors. Species differences have been

PAGE 34

24 previously noted with melanocortin receptors 42 and therefore further studies should be conducted with human subjects to confirm our results. In addition, structural differences distinguish the linear compound I 125 -NDP-MSH used by Tota et al. for binding activity assays and the cyclic compound I 125 MTII that we used for these assays. However, despite the differences in the radio-labeled compound used, we tested both I 125 -NDP-MSH and I 125 MTII at mMC1R, mMC3R, mMC4R, and mMC5R and found them to have similar IC 50 values at each receptor. Therefore, our binding assay results using I 125 MTII are comparable to previous research with either compound. Conclusion Researchers have proposed that the Arg-Phe-Phe sequence located in the C-terminus of agouti signal protein and AGRP is intimately involved in molecular recognition and antagonist activity of these molecules and the melanocortin receptors. The Arg-Phe-Phe sequence conserved in both of these compounds is structurally related to the conserved sequence His-Phe-Arg-Trp found in the melanocortin-stimulating hormone compounds. Furthermore, the Arg-Phe-Phe sequence may be mimicking the interactions of the sequence His-Phe-Arg-Trp with the melanocortin receptors. The results of this study provide evidence that supports this hypothesis.

PAGE 35

APPENDIX CHEMICAL STRUCTURES Table A-1 Chemical structures for FMOC amino acids O O N H O O H C H 3 C H 3 C H 3 Fmoc N H O O H C H 3 C H 3 C H Fmoc 3 Reagent Structure 9-fluorenyl-methoxy-carboxyl with (FMOC) Amino Acid Fmoc-Arg(Pbf) Fmoc-Asp(tBu) Fmoc-Tyr (tBu) 25

PAGE 36

26 Table A-1 Continued Reagent Structure Fmoc-Phe Fmoc-Cys (Trt) Fmoc-Ala Fmoc-Tyr(tBu)Wang resin O H O N H Fmoc O H N Fmoc O S C H 3 C H 3 C H 3 H C H 3 O O N H H Fmo

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27 Table A-2 Chemical structures for FMOC reagents Reagent Structure benzotriazol-1-yl-oxy-tris(dimethylamino phosphonium hexafluorophosphate (BOP) 1-hydroxybenzotriazole (HObt) Table A-3 Various reagents for Solid Phase Synthesis Benzotriazol-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP): 3-fold excess, 0.34g/amino acid N,N-diisopropylethylamine (DIEA) 5.1-fold excess, 218 L 10%: 50mL DIEA q.s. to 500mL with DMF 1-hydroxybenzotriazolemonohydrate (HOBt) (1M) (light sensitive): 27.2g HOBt q.s. to 200mL with DMF (FW= 135.13) 760 uL (1 mmol) N,N-dimethylformamide (DMF) (freshly distilled on sieves before use) 20% Piperidine in DMF (2:1) Kaiser Solution A 0.01M potassium cyanate (KCN) (33mg KCN q.s to 50mL H 2 O) added to 98mL Pyridine (bubbled with helium) Kaiser Solution B 2.5g Ninhydrin q.s. to 50mL with n-butanol Kaiser Solution C 80g phenol in 20mL n-butanol (leave in warm place overnight)

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REFERENCES 1. Flegal KM, Carroll MD, Ogden CL, Johnson CL. Prevalence and Trends in Obesity Among US Adults, 1999-2000. JAMA. October 9, 2002 2002;288(14):1723-1727. 2. Flegal KM, Graubard BI, Williamson DF, Gail MH. Excess Deaths Associated With Underweight, Overweight, and Obesity. JAMA. April 20, 2005 2005;293(15):1861-1867. 3. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD. Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature. Jan 9 1997;385(6612):165-168. 4. Jackson IJ. Homologous pigmentation mutations in human, mouse and other model organisms. Hum Mol Genet. 1997;6(10):1613-1624. 5. Cone RD. The central melanocortin system and its role in energy homeostasis. Ann Endocrinol (Paris). Mar 1999;60(1):3-9. 6. Fisher SL, Yagaloff KA, Burn P. Melanocortin-4 receptor: a novel signalling pathway involved in body weight regulation. Int J Obes Relat Metab Disord. Feb 1999;23 Suppl 1:54-58. 7. Hadley ME, Haskell-Luevano C. The proopiomelanocortin system. Ann N Y Acad Sci. Oct 20 1999;885:1-21. 8. Solomon S. POMC-derived peptides and their biological action. Ann N Y Acad Sci. Oct 20 1999;885:22-40. 9. Starowicz K, Przewlocka B. The role of melanocortins and their receptors in inflammatory processes, nerve regeneration and nociception. Life Sci. Jul 4 2003;73(7):823-847. 10. Fluck CE, Martens JWM, Conte FA, Miller WL. Clinical, Genetic, and Functional Characterization of Adrenocorticotropin Receptor Mutations Using a Novel Receptor Assay. J Clin Endocrinol Metab. September 1, 2002 2002;87(9):4318-4323. 28

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29 11. Butler AA, Cone RD. Knockout Studies Defining Different Roles for Melanocortin Receptors in Energy Homeostasis. Ann NY Acad Sci. June 1, 2003 2003;994(1):240-245. 12. Zhang L, Anthonavage M, Huang Q, Li W-H, Eisinger M. Proopiomelanocortin Peptides and Sebogenesis. Ann NY Acad Sci. June 1, 2003 2003;994(1):154-161. 13. Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Walson SJ, DelValle J, Yamada T. Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J. Biol. Chem. July 15, 1993 1993;268(20):15174-15179. 14. Tritos NA, Maratos-Flier E. Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone. Neuropeptides. Oct 1999;33(5):339-349. 15. Lu D, Willard D, Patel IR, Kadwell S, Overton L, Kost T, Luther M, Chen W, Woychik RP, Wilison WO. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature. Oct 27 1994;371(6500):799-802. 16. Miller MW, Duhl DM, Vrieling H, Cordes SP, Ollman MM, Winkes BM, Barsh GS. Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation. Genes Dev. Mar 1993;7(3):454-467. 17. Abdel-Malek Z, Suzuki I, Tada A, Im S, Akcali C. The Melanocortin-1 Receptor and Human Pigmentation. Ann NY Acad Sci. October 20, 1999 1999;885(1):117-133. 18. Rana BK. New Insights into G-Protein-Coupled Receptor Signaling from the Melanocortin Receptor System. Mol Pharmacol. July 1, 2003 2003;64(1):1-4. 19. Shutter JR, Graham M, Kinsey AC, Scully S, Luthy R, Stark KL. Hypothalamic expression of ART, a novel gene related to agouti, is upregulated in obese and diabetic mutant mice. Genes and Development. 1997;11(5):593-602. 20. Millhauser GL, McNulty JC, Jackson PJ, Thompson DA, Barsh GS, Gantz I. Loops and links: structural insights into the remarkable function of the agouti-related protein. Ann N Y Acad Sci. Jun 2003;994:27-35. 21. Bures EJ, Hui JO, Young Y, Chow DT, Katta V, Rohde MF, Zeni L, Rosenfeld RD, Stark KL, Haniu M. Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation. Biochemistry. 1998;37(35):12172-12177.

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30 22. Ollmann MM, Wilson BD, Yang Y-K, Kerns JA, Chen Y, Gantz I, Barsh GS. Antagonism of Central Melanocortin receptors in vitro and in vivo by agouti-related protein. Science. 1997;278(5335):135-138. 23. Willard DH, Bodnar W, Harris C, Kiefer L, Nichols JS, Blanchard S, Hoffman C, Moyer M, Burkhart W, Weiel J, Luther MA, Wilkison WO, Rocque WJ. Agouti structure and function: Characterization of a potent [alpha]-melanocyte stimulating hormone receptor antagonist. Biochemistry. 1995;34(38):12341-12346. 24. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. Jul 1 2003;31(13):3497-3500. 25. Tota MR, Smith TS, Mao C, MacNeil T, Mosley RT, Van der Ploeg LH, Fong TM. Molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors. Biochemistry. Jan 19 1999;38(3):897-904. 26. Merrifield R. Solid-Phase Peptide Synthesis: 3. An Improved Synthesis of Bradykinin. Biochemistry. September 1964 1964;14:1385-1390. 27. Stewart JM. Solid phase peptide synthesis. San Francisco: W. H. Freeman; 1969. 28. Carpino L, Han G. The 9-fluorenylmethoxycarbonyl amino-protecting group. Journal of Organic Chemistry. 1972;37:3404-3409. 29. Fields G. Solid-phase peptide synthesis. San Diego: Academic Press; 1997. 30. Graham M, Shutter JR, Sarmiento U, Sarosi I, Stark KL. Overexpression of Agrt leads to obesity in transgenic mice. Nat Genet. Nov 1997;17(3):273-274. 31. Stewart J, Young J. Solid phase peptide synthesis. Rockford, IL: Pierce Chemical; 1984. 32. Wang SS. p-alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments. J Am Chem Soc. Feb 21 1973;95(4):1328-1333. 33. Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem. Apr 1970;34(2):595-598. 34. Haskell-Luevano C, Shenderovich MD, Sharma SD, Nikiforovich GV, Hadley ME, Hruby VJ. Design, synthesis, biology, and conformations of bicyclic alpha-melanotropin analogues. J Med Chem. May 12 1995;38(10):1736-1750.

PAGE 41

31 35. Wigler M, Pellicer A, Silverstein S, Axel R, Urlaub G, Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. Mar 1979;76(3):1373-1376. 36. Jimenez A, Davies J. Expression of a transposable antibiotic resistance element in Saccharomyces. Nature. Oct 30 1980;287(5785):869-871. 37. Al-Obeidi F, Castrucci AM, Hadley ME, Hruby VJ. Potent and prolonged acting cyclic lactam analogues of alpha-melanotropin: design based on molecular dynamics. J Med Chem. Dec 1989;32(12):2555-2561. 38. Chen W, Shields TS, Stork PJ, Cone RD. A colorimetric assay for measuring activation of Gsand Gq-coupled signaling pathways. Anal Biochem. Apr 10 1995;226(2):349-354. 39. Schild HO. pA, a new scale for the measurement of drug antagonism. 1947. Br J Pharmacol. Feb 1997;120(4 Suppl):29-46; discussion 27-28. 40. Kiefer LL, Veal JM, Mountjoy KG, Wilkison WO. Melanocortin receptor binding determinants in the agouti protein. Biochemistry. 1998;37(4):991-997. 41. Haskell-Luevano C, Hendrata S, North C, Sawyer TK, Hadley VJ, Dickinson C, Gantz I. Discovery of prototype peptidomimetic agonists at the human melanocortin receptors MC1R and MC4R. J Med Chem. Jul 4 1997;40(14):2133-2139. 42. Mountjoy KG. The human melanocyte stimulating hormone receptor has evolved to become "super-sensitive" to melanocortin peptides. Molecular and Cellular Endocrinology. Jun 1994;102(1-2):R7-R11.

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BIOGRAPHICAL SKETCH Anzeela Mulaiya Schentrup was born in Mississauga, Ontario, Canada, to parents of Indian descent. She came to Florida at the age of 9 and grew up near Ft. Lauderdale. She first attended the University of Florida in 1994 and pursued a Bachelor of Arts degree in the history of science, in which she received highest honors for her thesis on pharmacy in Germany at the onset of the Industrial Revolution. Through exposure to the field of pharmacy sciences in the course of writing this thesis, Anzeela decided to pursue a scientific career leading to a graduate degree in medicinal chemistry. After two years, her scientific training stimulated her interest in a career in clinical medicine, so Anzeela left medicinal chemistry to pursue a Doctor of Pharmacy degree, which she received in 2004. During this time, she was encouraged by her former mentor, Carrie Haskell-Luevano, PhD, to pursue the Master of Science in medicinal chemistry for the work she had already completed. In addition, after the birth of her second child, and due to the faith of her current mentor, Julie Johnson, PharmD, Anzeela decided to return to graduate research in pursuit of the Doctor of Philosophy degree in pharmacogenomics. While in this degree program, Anzeela completed the Master of Science degree in medicinal chemistry and continues to pursue her doctorate. Although her educational background is diverse, all of the disciplines in which she has studied and developed a level of expertise have contributed to her current understanding of pharmacy practice and ability to perform translational research. Currently, Anzeela lives in Gainesville, Florida, with her husband, Joseph Ct, and two children, Ena Marie and Maximilian Ct. 32


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Title: Structure-Activity Relationships of an Agouti-Related Protein-Derived Decapeptide at the Murine Melanocortin Receptors
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Material Information

Title: Structure-Activity Relationships of an Agouti-Related Protein-Derived Decapeptide at the Murine Melanocortin Receptors
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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STRUCTURE-ACTIVITY RELATIONSHIPS OF AN AGOUTI-RELATED PROTEIN-
DERIVED DECAPEPTIDE AT THE MARINE MELANOCORTIN RECEPTORS
















By

ANZEELA MULAIYA SCHENTRUP


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Anzeela Mulaiya Schentrup

































This document is dedicated to my husband, Joseph, not only for being a loving,
supportive and dedicated partner, but for being mom and dad to our kids and to me when
I needed it. Any success I have is because of you.















ACKNOWLEDGMENTS

I owe thanks to many people for their role in the completion of this work. In

particular, I acknowledge Carrie Haskell-Luevano, PhD, my mentor for this project, for

her guidance and willingness to support me in my career choices. I hope always to have

a professional relationship with her, and I admire her accomplishments and dedication to

her work and students. Also, I thank Julie Johnson, PharmD, for encouraging me to see

this project through and for showing me my place in the field of pharmacy sciences. I

cannot say enough about how important her professional influence has been. I recognize

Margaret James, PhD, for allowing me this opportunity to complete this project. And

finally, my thanks go to Kenneth Sloan, PhD, for directing me into the exciting area of

pharmacy research in the first place and helping me with some of the most important

career decisions that I have made.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ......... .................................................................................... iv

LIST OF TABLES .................................. ....... ............. vii

LIST OF FIGURES ............ .......... ............................... viii

ABSTRACT .............. .................. .......... .............. ix

CHAPTER

1 BACKGROUND AND SIGNIFICANCE...................................................................

Focus on the M elanocortin System Components ........................................ ..............2
Solid Phase Peptide Synthesis: "FM OC" Chemistry...............................................6..

2 M ATERIALS AND M ETHODS ........................................ ........................... 9

Synthesis of the Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr Cyclic
D ecapeptide ...................................................... .. ........ .... .......... 9
Solid Phase Peptide Synthesis Preparation................................. .....................9
M annual Solid Phase Peptide Synthesis....................... ............... ............. ..11
Removal of Orthogonal Protecting Groups and Cleavage of the Peptide from
the R esin............................................ 13
Analysis of the Linear Peptide ......... ............... ............... ............... 14
Cyclization of the Linear Peptide.................... .. ............ .. ............... 15
A analysis of the Cyclized Peptide................... ......... ......... ........ .................... 15
B iolog ical A ssay s .................................................... ................ 15
C ell Culture and Transfection ........................................ ......... ............... 15
R eceptor B finding A says ............................................... .......... ............... 16
Quantification of the Receptor Binding Assays............... ................ 16
P -g alacto sidase B ioassay .................................................................................... 17
Quantification of P-galactosidase Bioassay .............. .......................................17
A analysis of B ioassay D ata .................................... ........................... ................. 18

3 R E SU L T S ....................... ..................................... ............... ...............19

R eceptor B finding .............................................................................................. 19
Receptor Antagonist Activity ................. ................................19









R eceptor A gonist A activity .................................................. .............................. 20

4 DISCUSSION AND CONCLUSION ........................................ ...................... 21

D isc u ssio n .............................................................................................................. 2 1
C conclusion ....................................................................................................... ........ 24

CH EM ICA L STRU CTURES ................................................... ............................... 25

REFERENCES ............................... .. .......... ...............28

B IO G R A PH IC A L SK E T C H ...................................................................... ..................32
















LIST OF TABLES


Table page

1-1 Melanocyte-stimulating hormone peptide structures ................................................2

2-1 A m ino acids used and quantities ........................................... ........................... 9

2-2 Protocol for manual FMOC chemistry synthetic strategy ............... ............... ....11

A-i Chemical structures for FM OC amino acids ................................... .................25

A-2 Chemical structures for FMOC reagents .............. .............................................27

A-3 Various reagents for Solid Phase Synthesis..................................... ............... 27















LIST OF FIGURES


Figure page

1-1 Alignment of Human ASP and Human AGRP in ClustalW24. The C-terminus is
in bold-face and the portion used to derive the decapeptide is indicated in italics.
The common Arg-Phe-Phe (RFF) motif is shaded .................................................5

2-1 This glass fritted filter glass container contains a stem with a bidirectional valve.
The filter flask is attached to a ring stand and placed above and connected to a
waste flask. The bidirectional valve is connected to a nitrogen gas line and the
waste flask is attached to a vacuum source. ....................................................10

2-2 An example of the deprotection scheme using FMOC chemistry. R = resin bead. ... 12

2-3 Activation of the coupling reaction by BOP. ........................................ ..................13

2-4 Cleavage of the peptide chain from the resin bead by TFA.................. ........... 14

3-1 P-galactosidase expression is given for varying concentrations of decapeptide with
the varying concentrations of M TII. ............................... .. ......................... 20

3-2 P-galactosidase expression is given for varying concentrations of decapeptide
peptide concentration. For comparison, the P-galactosidase expression for MTII
is giv en .............................................................................20















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

STRUCTURE-ACTIVITY RELATIONSHIP OF AN AGOUTI-RELATED PROTEIN-
DERIVED DECAPEPTIDE AT THE MARINE MELANOCORTIN RECEPTORS


By

Anzeela Mulaiya Schentrup

December 2005

Chair: Carrie Haskell-Luevano
Major Department: Medicinal Chemistry

Obesity is a complex, multi-factorial chronic disease involving several contributing

factors. The melanocortin neurohormone messenger system is thought to play a role in

modulating the obesity phenotype. In particular, Agouti-related protein (AGRP), an

endogenous antagonist of melanocortin receptors, interacts with the human brain

melanocortin receptors hMC4R and hMC3R. When AGRP is over-expressed centrally in

transgenic mice, the result is an obese phenotype. Thus, AGRP expression in the

hypothalamus is thought to be involved in the regulation of energy homeostasis at the

brain melanocortin receptors. We hypothesized that the AGRP antagonist activity is

determined by the structure-function relationship between specific amino acids in AGRP

and its receptor.

The decapeptide, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr, which is derived

from the AGRP C-terminal domain, has been reported to possess [tM antagonistic

properties at the hMC4R. We synthesized this decapeptide using a manual solid-phase









peptide synthetic strategy. In addition, we pharmacologically characterized this

decapeptide at the murine melanocortin receptors, mMC 1R, mMC3R, mMC4R and

mMC5R. In particular, we used the competitive displacement of I125-MTII, a radio-

labeled melanocortin agonist, to determine binding and the P-galactosidase bioassay to

determine receptor activity.

We found the decapeptide, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr, to be a

weak antagonist at the mMC4R (pA2 = 6.79) and to possess agonist activity (2.89 + 2.26

[M) at the skin mMC1R. Further, the decapeptide did not have appreciable activity at

the mMC3R or mMC5R. The agonist activity at the mMC1R skin receptor was

unexpected and contrasts with a previous report that AGRP has no activity at the MC1R.

Our study provides experimental evidence that the C-terminal portion of AGRP contains

a specific recognition sequence for melanocortin receptor activity. Furthermore, we

conclude that the conformational shape which the AGRP protein attains is a significant

determinant of AGRP activity at the melanocortin receptors.














CHAPTER 1
BACKGROUND AND SIGNIFICANCE

Obesity is a highly prevalent chronic disease in humans. Based on data from the

National Health and Nutrition Examination Survey (NHANES) from 1999-2002, 30

percent of adults 20 years of age and older (greater than 60 million people), had a body

mass index (BMI) of 30 or greater. This is significantly increased from 23 percent in

1994.1 The high prevalence of obesity gives rise to increasing numbers of patients with

obesity-related complications such as musculoskeletal disorders, cardiovascular disease,

decreased respiratory function and diabetes mellitus. These sequelae have a significant

negative impact on patients' quality of life as well as on health care cost. Furthermore,

because of these serious complications, obesity is now considered the second leading

cause of preventable death in the U.S. 1, 2 Current research on controlling obesity in

humans involves focus on the factors involved in obesity development.

Several factors, including genetic make-up, physiology, environment, behavior and

individual psychology all modulate obesity risk and severity. In particular, previous

studies identify the function of neuroendocrine messenger systems such as the

melanocortin system as a genetic and physiological factor which plays an important role

in human weight homeostasis in both animal models and humans3-6. Specifically, the

melanocortin messengers interact with several other central and peripheral neurohormone

messengers to affect weight homeostasis by modifying feeding behavior and

thermogenesis. By characterizing the key structural elements of the components of the

melanocortin system, researchers hope to uncover a powerful therapeutic target for drug









therapy in an effort to control obesity in humans. The following is a brief overview of the

melanocortin system and how it is purported to fit into the complex system of central

weight regulation.

Focus on the Melanocortin System Components

The melanocortin system consists of melanotrophic peptides, endogenous peptide

inhibitors and receptors. In particular, the melanotrophic peptide hormones are derived

from the proopiomelanocortin (POMC) gene transcript. POMC is the source of multiple

hormones including adrenocorticotropin (ACTH), B-lipotropin, met-enkephalin and P-

endorphin7. The POMC product that is produced at a given time depends on the hormone

system that activates POMC transcription. When POMC is activated and transcribed as

part of melanocortin activation, it gives rise to a-, y- and p- melanocyte stimulating

hormone (Table 1-1). Most research has focused on a-melanocyte stimulating hormone

Table 1-1 Melanocyte-stimulating hormone peptide structures
Chemical Sequences
a-MSH Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
13-MSH NH2-Asp-Glu-Gly-Pro-Tyr-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Pro-
Lys-Asp-OH
y-MSH NH2-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH


(a-MSH), a 13-amino acid peptide (Figure 1-1) which is the prototype endogenous

agonist of the melanocortin system receptors. It is produced both centrally and

peripherally and has been found to stimulate the five melanocortin receptor subtypes.

The melanocortin receptors are G-protein coupled receptors (GPCRs). They have

the classic GPCR secondary structure consisting of seven lipophilic a-helical membrane-

spanning regions. The N-terminus is exposed on the extracellular portion of the

membrane while the C-terminus remains intracellular. In this construct, the melanocortin

peptides putatively interact with the helical pocket that is formed by the surrounding









transmembrane regions of the receptor. Interaction of the ligand and receptor induces a

conformational change in the target receptor resulting in activation of the receptor.

Activated melanocortin receptors interact with a trimeric G-protein. In the case of

the melanocortin receptors, this interaction stimulates Gs which activates adenylate cylase

(AC). AC triggers an intracellular cascade of signaling in which adenosine triphosphate

(ATP) is converted to cyclic adenosine monophosphate (cAMP). Cyclic AMP then acts

as a second messenger and modulates subsequent physiological activity7. Specifically,

melanocortin receptor activation causes an increase in cAMP-mediated phosphokinase A

(PKA) activity which activates the cAMP response element binding protein (CREB).

Then, CREB interacts with cAMP response element (CRE) on deoxyribonucleic acid

(DNA) to activate cell-specific transcription of mRNA resulting in a physiological

response.

Five subtypes of melanocortin receptors in several species have been identified and

are numbered in the order in which they were originally cloned. Further, murine receptor

isolates are homologous to human receptors8. The melanocortin 1 receptor (MC1R) is

located peripherally in both humans and mice and is found in melanocytes, melanoma

cells and macrophages. MC1R is known to affect pigmentation and are reputed to be

involved in the inflammatory response9, however all of the mediators and specific effects

of MC1R involvement are as yet unknown. The melanocortin 2 receptor (MC2R) is

responsive only to ACTH and is located in all three layers of adrenal cortex as well as on

adipocytes. These receptors are involved in adrenal steroidigenesis and also have

adrenocorticotrophic effectso. The melanocortin 3 receptor (MC3R) is expressed in

various areas, particularly in the brain, including the arcuate nucleus and the anterior









ventral periventricular nucleus. In addition, the MC3R is expressed in the heart, placenta,

pancreas and gut. MC3R, like the melanocortin 4 receptor (MC4R), discussed below,

plays a role in weight homeostasis1. The melanocortin 5 receptor (MC5R) is expressed

ubiquitously in muscle, liver, spleen, lung, brain, adipocytes, and a variety of other

tissues. Deletion of MC5R in animal experiments results in exocrine gland dysfunction,

however more research regarding its specific function is needed2.

The MC4R is primarily expressed in the CNS, specifically in cortex, hippocampus,

thalamus and hypothalamus 13. In terms of function, MC4R is involved in feeding

behavior and weight homeostasis in both animal and human models. MC4R knockout

mice develop obesity, hyperphagia, hyperinsulinemia and hyperglycemia, indicating that

receptor activation is important in inducing satiety and preventing obesity4.

The melanocortin system is currently the only GPCR system that is known to

involve both endogenous receptor agonists and antagonists. In particular, the agouti gene

locus (ASIP) encodes Agouti Signaling Protein (ASP), a known antagonist of the

melanocortin receptors in animal models15. This protein is expressed in the periphery and

is responsible for the phenotype of the lethal Ay mouse, in which ASP is ectopically

expressed. Phenotypically, lethal Ay mice are obese with a yellow coat color16 as a result

of this ectopic ASP expression. The yellow coat color is a result of a shift in production

of eumelanin to pheomelanin17 that occurs as a result of ASP interaction with MC1R.

Also, ASP antagonizes central melanocortin receptors in lethal Ay mice, leading to the

obesity trait. Although this antagonism occurs centrally, ASP is not known to be

expressed endogenously in the central nervous system. 18 Researchers have used this









result to look for a protein analogous to ASP that is produced centrally and may

contribute to the obese phenotype in mice and humans.

Homology studies with ASP in several species lead to the discovery of Agouti-

Related Protein, or AGRP, which is a centrally-expressed analog to ASP in both murine

and human models19. ASP and AGRP are 131-amino-acid proteins in mouse and 132-

amino-acid proteins in humans. Further, both have cysteine-rich C-termini with

secondary structure that is maintained through disulfide bridges19-21. The sequence

homology between ASP and AGRP is 40% percent in the C-terminus19 (Figure 1).

AGRP is known to bind and antagonize both MC3R and MC4R, while ASP is active at

both MC1R and MC4R15 2223. Previous research has identified the triplet of amino acids

Human ASP MDVTRLLLATLLVFLCFFTANSHLPPEEKLRDDRSLRSNSSVNLLDVPSV 50
Human AGRP -----MLTAAVLSCALLLALPATRGAQMGLAPMEGIRRPDQALLPELPGL 45

SIVALNKKSKQIGRKAAEKKRSSKKEASMKKVVRPRTPLSAP-CVATRNS 99
GLRAPLKKTT--AEQAEEDLLQEAQALAEVLDLQDREPRSSRRCVRLHES 93

CKPPAPACCDPCASCQCRFFRSACSCRVLSLNC------ 132
CLGQQVPCCDPCATC YCRFFNAFCYCRKLGTAMNPCSRT 132

Figure 1-1 Alignment of Human ASP and Human AGRP in ClustalW24. The C-terminus
is in bold-face and the portion used to derive the decapeptide is indicated in
italics. The common Arg-Phe-Phe (RFF) motif is shaded.

at positions hAGRP (111-114), Arg-Phe-Phe, as necessary for MC4R antagonism.25

Further, structural studies indicate that these residues form an active loop region in the

peptide which interacts with the melanocortin receptors20. Short cyclic fragments of the

C-terminus of ASP and AGRP that contain the Arg-Phe-Phe domain were shown by Tota

et al to possess potent antagonist activity at MC4R but 10-fold lower activity and binding

than that of the full C-terminus25. In addition, no activity was seen for these fragments at

MC3R.









The purpose of the current study is to identify binding interactions between

synthetic melanocortin receptor antagonists and the melanocortin receptors in order to

logically design molecules to modulate the activity of melanocortin receptors and also to

provide information about the structure of these receptors and antagonists in vivo.

Specifically, this study looks at the interaction of a synthetic cyclic decapeptide derived

from AGRP with all five murine melanocortin receptors. A brief overview of the

synthetic strategy used to synthesize the test compound follows. The specific synthetic

scheme is given in the Methods section of Chapter 2.

Solid Phase Peptide Synthesis: "FMOC" Chemistry

Solid-phase peptide synthesis was conceived by R.B. Merrifield in 195926. The

strategy involves the covalent attachment of a nascent peptide chain to an insoluble

polymeric support resin. The resin is maintained in a filter system that allows the peptide

to be anchored and easily separated from reaction reagents. This procedure allows the

use of excess soluble reagents which can be filtered and washed from the reaction vessel

once amino-acid coupling reactions have terminated. The use of excess reagents allows

the reactions to be driven to high yields (>90%). In addition, the catalysis of these

reactions minimizes the formation of side products. By using a series of deprotection and

coupling steps, the researcher can select specific reactive moieties on the peptide to

participate in chemical reactions while others remain protected. Since this rationally

designed system of synthesis is generous in allowing high concentrations of reagents and

the synthesized molecule is easily separated from reagents, the system lends itself well to

automation27

Synthetic methods of solid phase peptide synthesis are differentiated based on the

protection scheme used. Protection schemes require the use of both N-a- and side chain









protection moieties so that the exposed reactive portions of the nascent peptide can be

manipulated separate from each other. During peptide synthesis, temporary N-a-

protecting groups are removed with each amino-acid coupling step to reveal the portion

of the amino acid which will be coupled to the next residue. Permanent side chain

protecting groups are used to protect vulnerable side chain groups during coupling

reactions and are removed altogether after synthesis has been completed.

The "FMOC" synthetic strategy was used to synthesize the test compounds in this

study. This strategy is based on an orthogonal system of protecting groups developed by

Carpino and Han28. Orthogonal schemes are based on using two or more classes of

protecting groups that can be removed by differing chemical mechanisms. FMOC

chemistry utilizes molecules constructed using a base labile "temporary" Na-9-

fluorenylmethyloxycarbonyl (FMOC) group. The permanent protecting groups are

primarily based on tertiary-butanol esters and ethers which provide bulky, base-stable

protection to otherwise labile amino acid side chains. During synthesis, the temporary

FMOC group is removed sequentially after each amino acid is added in order to form the

growing peptide chain while the permanent protecting groups remain intact until the end

of synthesis29

The FMOC synthetic scheme described above is used in this study to produce

synthetic peptides for use in research on the melanocortin system. In particular, this

study is focused on Agouti-related protein (AGRP), a component of the melanocortin

system that is of particular interest as an obesity target. In particular, previous research

establishes that AGRP participates in normal signaling for human feeding behavior and

energy homeostasis30. Therefore, structural alterations in AGRP may affect this normal









signaling and thereby contribute to the development of obesity. To structurally

characterize the activity of AGRP at the melanocortin receptors, biochemists have

isolated the cyclic decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr from the

AGRP C-terminus25. According to Tota et al., this small portion of the AGRP molecule

binds to and antagonizes the human MC4R. In order to verify this finding which

establishes the cyclic decapeptide as the active portion of AGRP and further analyze its

interaction with melanocortins receptors that were previously unstudied, we synthesized

the decapeptide using manual solid phase peptide synthesis using the FMOC strategy

described by Stewart et al. 31. We then pharmacologically characterized the peptide at the

5 murine melanocortin receptors. We used the murine model of melanocortin receptor

action to test our hypothesis that the cyclic decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-

Phe-Cys]-Tyr is the minimally active fragment of the AGRP molecule and will thus have

antagonist properties at the mMC3R and mMC4R. Further, the decapeptide will not have

significant activity, similar to its parent AGRP, at other murine melanocortin receptors.














CHAPTER 2
MATERIALS AND METHODS

The synthesis of the cyclic decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-

Tyr was accomplished by manual solid phase peptide synthesis using the FMOC

synthetic strategy and then tested in vitro for binding and activity at the murine

melanocortin receptors.

Synthesis of the Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr Cyclic Decapeptide

Solid Phase Peptide Synthesis Preparation

Reaction reagents and their structures are listed in the Appendix and corresponding

reaction quantities are shown in Table 2-1. All peptide reagents and resin were obtained

from Peptides International (Louisville, KY, USA). All reagents and solvents were

American Chemical Society (ACS) grade or higher.

Table 2-1 Amino acids used and quantities
Amino Protection Equivalents Molar quantity Molecular Amount (g)
Acid Scheme (mmol) Weight
Cys FMOC/Trt 3.13 0.76 585.73 0.45
Phe FMOC 3.13 0.76 387.44 0.29
Ala FMOC-H20 3.13 0.76 329.36 0.25
Asn FMOC/Trt 3.13 0.76 596.69 0.45
Arg FMOC/Pbf 3.13 0.76 648.78 0.49
Tyr FMOC/tBu 3.13 0.76 459.55 0.35

The manual FMOC synthesis was conducted in a reaction vessel consisting of a

glass-fritted filter and a valve separating two ports. First, the glass reaction vessel was

acid-washed with chromerge and concentrated H2SO4. The vessel was then silanized

using 10% dichloromethylsilane (DCMS) in dry toluene and mixed using helium gas for









15 to 30 minutes. After mixing, the reaction vessel was rinsed with toluene, then treated

with dry methanol for 15 minutes, and then dried with acetone.

Upon assembly of the reaction apparatus, one port of the reaction vessel was

attached to nitrogen gas and the other to a vacuum line for waste removal. The reaction

vessel was then mounted to a ring stand inside a vertical laminar flow hood. A waste

flask was secured beneath the reaction vessel (Figure 2-1). The gas and vacuum lines

were primed.










t to Vacuum





Figure 2-1 This glass fritted filter glass container contains a stem with a bidirectional
valve. The filter flask is attached to a ring stand and placed above and
connected to a waste flask. The bidirectional valve is connected to a nitrogen
gas line and the waste flask is attached to a vacuum source.

FMOC-Wang resin32 beads (Appendix) were added to the pre-weighed reaction

vessel and weighed. Since the dry polystyrene resin beads have an average diameter of

50 rim, the beads were then soaked in dimethylformamide (DMF) for two hours to

promote swelling. After this process, the beads obtained 2.5 to 6.2 times their dry

volume and formed a well-solvated gel. This maximally increases the bead surface area

and exposes the reagent-accessible chemical attachment points.









Manual Solid Phase Peptide Synthesis

The synthesis procedure is outlined in Table 2-2. After copious rinsing of the
Table 2-2 Protocol for manual FMOC chemistry synthetic strategy
Cycle Function Time
1 DMF wash 5 X Imin
2 20% Piperidine in DMF rinse 2 min
3 20% Piperidine in DMF deprotection 18 min
4 DMF wash 4 X 1 min
5 Ninhydrin Test 3 min
6a-d Add : FMOC-AA dissolved in DMF (3 eq.)
BOP (3 equiv.)
HOBt (3 equiv.)
DIEA (5.1 equiv.)
7 Coupling
8 Ninhydrin Test 3 min
9 DMF wash 3 X 1

solvated resin with DMF, the beads were rinsed with 20% piperidine for 2 minutes,

drained, and then mixed again for 18 minutes in 20% piperidine to remove the FMOC

protecting group (Figure 2-2). The resin was then rinsed with DMF and the Kaiser

ninhydrin test 33 (described below) was conducted to observe the extent of deprotection.

For the ninhydrin test, a small sample of the resin was removed by capillary pipette

and placed into a small glass vial. Two drops of each of the Kaiser ninhydrin test

solutions A, B and C (Appendix) were added to the vial. The vial was then heated to

approximately 1000C for 3 minutes. A positive test indicating complete deprotection of

the free peptide chain amino group was given by a homogeneous deep blue color. If a

negative test was obtained, the piperidine deproctection step was repeated until the

ninhydrin test was positive.











N -9-florenylmethyloxycarbonyl
protecting group (FMOC)
Fmoc-Tyr(tBu)-Wang resin
Tyrosine (Tyr)


0H 0 O

H o Glycine (Gly)

Piperldme H CH
o CH3
Aspartic Acid (Asp) HC





Fmoc-Tyr(tBu)-Wang resin
Free Amino Group o
/ H\ HN NH NH o o

CH2 o-= 0

H3C-CH3
0==C=O CH3 0 CH
Carbon Dioxide Gas H/
CH3


Figure 2-2 An example of the deprotection scheme using FMOC chemistry. R = resin
bead.

After confirmation of successful deprotection, the next amino acid in the

decapeptide sequence was added to the reaction vessel in the amount given in Table 2.1.

In addition, benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate

(BOP) in 3-fold excess (Appendix) was added to accelerate the formation of the -Obt

ester form of the FMOC amino acids (Figure 2-3). Also, 1-hydroxybenzotriazole (HOBt)

(Appendix) in 3-fold excess was used for three reasons: (1) to accelerate the

cabodiimide-mediated couplings, (2) to suppress racemization and (3) to inhibit

dehydration of the carboxamide side chains of Asn and Gln to the corresponding nitriles.

Finally, N,N-diisopropylethylamine (DIEA) in 3.1 fold excess was used to stabilize side

products of the amino acid coupling reaction (Figure 2.3). Completion of the amino acid















BOP
benzotriazol-1 -yl-oxy-tris(dimethylammno)
phosphonum hexafluorophosphate




N \\
F
\ F IF

(H3C)2N /O F

FMOC-N
(H3C)2N N(CH3)2


H3C
O-CH-
-HN Tertiary Amme Base
H3C --CH3
H3C
DIEA
Dixsopropylethylamine


0
FMOC -NH o


R,

Fmoc Amino Acid


o N(CH 3)2
P' FMOC -NH ,
(H3C)2N/ I N(CH3)2 +
OBt
R


N -OBt
" HOBt
S/ DIEA


O
H

O \/ N(CH 3)2
R \

(H3C)2N N(CH3)2




tN\\N
N
-OBt \


DEA


FMOC -NH

O +
R,
"t


HOBt-active ester


H2N-R

Growing Deprotected Peptide



0
FMOC -NH

N NH
R1

Coupled Peptide









NH-FMOC
Symmetric Anhydride
R/


hexamethylphosphoramide /N(CH 3)2

(H3C)2N/ "N(CH3)2

Figure 2-3 Activation of the coupling reaction by BOP.


addition and FMOC deprotection was monitored using the ninhydrin test. After repeating


the procedure nine times to complete the linear peptide Tyr-Cys-Arg-Phe-Phe-Asn-Ala-


Phe-Cys-Tyr, the reaction vessel containing the resin and newly formed peptides was


dried under vacuum overnight. The weight of the peptides and resin combination was


determined after drying was complete.


Removal of Orthogonal Protecting Groups and Cleavage of the Peptide from the
Resin


The orthogonal protecting groups on the peptide amino acids were cleaved from the


peptide in the same step as the peptide cleavage from the resin beads. This was


accomplished using a trifluoroacetic acid (TFA), 1,2 ethanedithiol, p-cresol, and water


cleavage cocktail in a 15:3:1:1 ratio (Figure 2-4).











Linker +
Tyrosine(Tyr) Resin

HO NH NHy NHO


Glycine (Gly)
S

H3C CH3
CH3 O
Cysteine(Cys) CH3
H3C CH3




OH

F Trifluoroacetic Acid (TFA)


Figure 2-4 Cleavage of the peptide chain from the resin bead by TFA.

Five milliliters of the cleavage cocktail were added to a medium round bottom flask with

the dried resin and allowed to mix for 2 hours at 0C and then 30 to 45 minutes at room

temperature. The decapeptide was then separated from the resin by filtration and the

resulting solution was concentrated. After concentration, the peptide was extracted using

water and ethyl ether and the extract was concentrated until a white flocculent appeared.

The resulting crude peptide was then lyophilized.

Analysis of the Linear Peptide

The success of the manual linear peptide synthesis was confirmed using high

performance liquid chromatography (HPLC) purification with a Shimadzu

chromatography system with a photodiode array. The structure was confirmed with gas

chromatography/ mass spectroscopy (GC/MS).









Cyclization of the Linear Peptide

Upon confirmation of the weight of the linear peptide by analytical methods, a

disulfide bridge was formed34 between the two cysteine residues on the linear

decapeptide Tyr-Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys-Tyr. Half of the crude peptide was

dissolved in 1.5 L 0.1% degassed acetic acid in water. The pH was adjusted from 3.3 to

8.5 with concentrated ammonium hydroxide and the solution was then mixed overnight.

After mixing, the pH of the solution was lowered to 4.0 with glacial acetic acid. A non-

measured amount of HCl-amber-lite ion exchange resin was added and this combination

was mixed until the yellow solution turned clear (-1 hour). The exchange resin was then

filtered into a large round bottom flask and concentrated. The resulting compound, Tyr-

[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr was lyophilized.

Analysis of the Cyclized Peptide

The final cyclized product was analyzed by MALDI-TOF mass spectroscopy at

the University of Florida Protein Core Facility. Final peptide purification was achieved

using a semi-preparative RP-HPLC Cis- bonded silica column (Vydac 218TP1010, 1.0

X 25 cm). The structure was assessed by analytical RP-HPLC and 2D H NMR.

(University of Florida protein core facility).

Biological Assays

Cell Culture and Transfection

HEK-293 cells (human embryo kidney cells transformed with human adenovirus

type 5) were maintained in Dulbecco's modified Eagle's medium (DMEM), 10%

penicillin-streptomycin and 10% fetal calf-serum. The cells were seeded 1 day prior to

transfection at 1-2 x 106 cells/100mm. Murine melanocortin receptor cDNA (coding

deoxyribonucleic acid) (20 [tg) cloned into in the pcDNA3 expression vector in a









previous experiment was transfected into the seeded HEK-293 cells using the calcium

phosphate method35. G418 sulfate aminoglycoside (C20H40N4010.2H2SO4) selection36

was used to isolate stable receptor populations (1 g/mL).

Receptor Binding Assays

HEK-293 cells that were transfected and were stably expressing the various

melanocortin murine receptors (mMC1R, mMC3R, mMC4R and mMC5R) were

maintained in Dulbecco's modified Eagle's medium (DMEM), 10% penicillin-

streptomycin and 10% fetal calf-serum. One day prior to conducting the binding studies,

transfected cells were plated into Primera 24 well plates (Falcon) at a density of 0.1-0.3 x

106 cells perwell. Concentrations of 10-6 to 10-12 M MTII (Ac-Nle-c[Asp-His-D-Phe-

Arg-Trp-Lys]-NH2), a potent melanocortin receptor agonist37, and 10-4 to 10-10 M Tyr-

[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr were used to competitively displace MTII that

was radio-labeled with 1125 (100000 cpm/well; NEN Life Sciences). A 450 pL solution of

the concentration of decapeptide being tested was added to each well. Next, 50 PL of 1125

MTII was added to each well and incubated at 370C for 1 hour. The medium was then

rinsed and washed with assay buffer (DMEM, 0.1 mg/ml and Bovine Serum Albumin

(BSA)). The cells were lysed with 0.5 mL 0.1 M NaOH and 0.5 mL 1% Triton X-100 for

10 minutes, and then transferred to 16 X 150 mm glass tubes.

Quantification of the Receptor Binding Assays

The binding of I125-radiolabeled MTII was measured using a y-counter. Dose-

response curves and IC5o values for 10-6 to 10-12 M MTII and 10-4 to 10-10 M Tyr-[Cys-

Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr were generated and analyzed by nonlinear least-

squares analysis. The IC5o values represent the mean (+ standard deviation) of duplicate

wells generated in at least two independent experiments.









p-galactosidase Bioassay

HEK-293 cells stably expressing melanocortin receptors (mMC1R, mMC3R,

mMC4R, mMC5R) were transfected with 4 pg of the CRE/p-galactosidase reporter

gene38 using the same transfection method described above. Primera 96-well plates were

treated with 5000-15000 post-transfection cells and incubated overnight. Forty-eight

hours post-transfection, cells were stimulated with MTII (with 10-6 to 10-12 M), Tyr-[Cys-

Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr (10-4 to 10-10 M) and forskolin (10-4 M) in assay

medium (DMEM containing 0. Img/mL BSA and 0.1 mM isobutylmethylxanthine) for 6

hours. The assay medium was aspirated and 50 iL of lysis buffer (250 mM TrisHC1, pH

8.0, and 0.1% Triton X-100) was added to each well. The plates were then stored at -

800C overnight. The next day, the plates containing the cells lysates were thawed andl0

[IL aliquots were taken from each well and transferred to another 96-well plate. The 10

[IL samples were set aside for relative protein determination. Phosphate-buffered saline

(PBS) with 0.5% BSA (40 [iL) was then added to each well of the original plates of cell

lysate. Further, 150 iL of substrate buffer (60 mM sodium phosphate, 1 mM MgC12, 10

mM KC1, 5 mM 3-mercaptoethanol, 200 mg/mL 2-Nitrophenyl-P-D-galactopyranoside

(ONPG)) was added to each well and the plates were incubated at 370C.

Quantification of P-galactosidase Bioassay

The sample absorbance OD405 was measured using a 96-well plate reader. The

relative protein value was determined by adding 200 pL 1:5 dilution of G250 protein dye

(Bio-Rad):water to the 10 tL cell lysate aliquots taken previously. In addition, the OD595

was measured. Furthermore, the transfection efficiency of the CRE-p-galactosidase

reporter assay was determined using 10-4 M forskolin treatments of 6 wells of each plate

as controls. Data points were normalized both to the relative protein content and non-.






18


Analysis of Bioassay Data

Data analysis was conducted and IC50 and EC50 values were determined by using

nonlinear regression analysis with the PRISM program (v2.0, GraphPad Inc.). In

addition, pA2 values quantifying antagonism were generated using the Schild analysis

method.39 The EC50 and pA2 values represent the mean (+standard deviation) of triplicate

wells examined in at least two independent experiments.














CHAPTER 3
RESULTS

The synthesis of the melanocortin decapeptide, Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-

Phe-Cys]-Tyr, was conducted with a yield of 0.5g of product. We confirmed that each of

the coupling steps in the synthesis was completed using the ninhydrin test as previously

described. The purified peptide was > 99% pure and the sequence was determined by

methods described previously to be the sequence of the desired decapeptide with the

correct molecular weight.

Both the binding activity and function of the decapeptide at the murine

melanocortin receptors was determined.

Receptor Binding

The hAGRP decapeptide bound extensively to the mMC4R (IC50 = 275 62 nM).

Additionally, the decapeptide possessed [aM binding IC50 values at mMC3R (IC50 = 11.7

+ 3.9 [M), mMC5R (IC50 = 38.7 23.5 gM), and mMC1R (IC50 = 3.09 2.19 [M).

Receptor Antagonist Activity

The hAGRP decapeptide was an antagonist of mMC4R (pA2 = 6.8 0.4) (Figure

3-1). In addition, it has only slight antagonist activity at the mMC3R which was not

significant enough to quantify, and lacked antagonist activity at mMC5R at

concentrations up to 100 [M.














Receptor Agonist Activity


The hAGRP decapeptide was found to have agonist activity at mMC1R (EC5 =


2.89 + 2.26 [M) (Figure 3-2).. No agonist activity was detected for the decapeptide at


any of the other melanocortin receptors (mMC3R, mMC4R, mMC5R).


mMC3R


* MTII
A 100OnMYC[CRFFWNFCJY MTI
V 1000nM YCCRFFNAFCJY + ITII
* 100nMYCIjRFFNFCfv nMT
* IOnM Yc(CFFM FCY* MTI
o YdCRFFNAFClY


13 -12 -11 -10 -8 -7 -6 -5 -4 -3
Log MTII Concentration (M)


1 O5
1,25
S100
0 75
050
25 oso
025


mMC5R


I F MTII
S f[3 Yc(CRFFNAFCjY


n-n~D--n-nc-


00041-- -- .-
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -
Log Peptide Concentration


mMC4R
1.25- h MlT
A IOOOOMYC(CRFFNAFC]Y MFTI
1O- i0 00OnM Yc(CRFFNAFCIY + MTII
100nM YcCRFFNAFCJY +MTII
0 75- 10nM Yc(CRFFNAFC]Y MTII
050 YClCRFFNAFCIY

0 25 i o- i

-13 -12 -11 -10 -9 -8 -7 6 -5 *4 -3
Log Peptide Concentration (M)


Figure 3-1 P-galactosidase expression is given for varying concentrations of decapeptide

with the varying concentrations of MTII.


mMC1R


U MTII
A Yc[CRFFNAFC)Y


-13 -12 -11 -10 -9 -8 -7 -4 -3
Log Peptide Concentration (M)

Figure 3-2 j-galactosidase expression is given for varying concentrations of decapeptide

peptide concentration. For comparison, the j-galactosidase expression for

MTII is given.


1.25-

. 100 ,
1I,

B 075

050

0.25-

000














CHAPTER 4
DISCUSSION AND CONCLUSION

Discussion

Our most significant finding in this study is that the AGRP decapeptide Tyr-[Cys-

Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr has both unexpected agonist activity at the mMC1R

while also being an antagonist of the mMC4R. The antagonist activity at mMC4R that

we observed is consistent with our hypothesis and previous research30, however, the

agonist activity at mMC1R has not been described in previous studies. Furthermore, our

findings imply that the conformational space that AGRP occupies in vivo is related to the

activity of AGRP at the melanocortin receptors.

The finding that the AGRP decapeptide has activity at the mMC1R is in contrast

with previous research which has shown that the parent AGRP protein has no activity at

the skin mMC1R22. These previous studies are supported by the finding that transgenic

mice ectopically expressing AGRP have the brown (wild-type) coat phenotype. This

previous result indicates that AGRP does not shift the production of eumelanin to

pheomelanin in these transgenic mice and, thus, AGRP has no activity at skin mMC1R.

In our study, we also expected to find that the AGRP decapeptide that we studied would

have no agonist activity at the mMC1R. However we observed that the AGRP

decapeptide has micromolar agonist activity at the mMC R and sufficient binding at this

receptor (3.09 + 2.19 aM) to explain this activity as a direct receptor effect.

Since the AGRP decapeptide amino acid sequence is derived from the sequence of

its parent protein, AGRP, the contrasting activity between the decapeptide and its parent









peptide at the mMC 1R cannot be explained by the decapeptide amino acid sequence

alone. Specifically, the decapeptide possesses the identical amino acid sequence as the

active portion of the human and murine AGRP C-terminus (108-177)25. Although the

decapeptide has some distinct chemical moieties in analogous positions of the human and

murine Agouti Signal Protein (Figure 4.1), the decapeptide retains the Arg-Phe-Phe motif

in a conserved position between AGRP and ASP. This Arg-Phe-Phe sequence is thought

to be essential for the antagonist activity of the melanocortin antagonists40. Moreover, in

contrast to AGRP, when mASP is ectopically expressed in transgenic mice, mice with the

agouti coat color are produced. The resulting agouti coat color indicates that mASP is an

antagonist at the skin mMC1R41. Thus, although the AGRP decapeptide sequence is

derived from a consensus sequence of the AGRP and ASP C-terminus, the AGRP

decapeptide itself has different pharmacological activity than both of its parent proteins.

Previous research has indicated that the conserved Arg-Phe-Phe (RFF) motif

found in both the ASP and AGRP active sites may be important for receptor recognition

of these endogenous antagonists. In support of this idea, studies have shown that

mutation of these Arg-Phe-Phe residues in AGRP results in considerably less efficacious

antagonism at the melanocortin receptors.4 Furthermore, homology exists between the

Arg-Phe-Phe motif found in the melanocortin endogenous antagonists and the conserved

His-Phe-Arg-Trp residues in the endogenous melanocortin ligands. Specifically, the Arg-

Phe-Phe motif may be mimicking the His-Phe-Arg-Trp molecular interactions and

allowing the melanocortin receptors to recognize AGRP and ASP. Moreover, previous

studies have found that the tetrapeptide Ac- His-Phe-Arg-Trp -NH2 is the minimal

fragment of melanocortin agonists required to produce a physiological response (pM) in









the classic frog skin bioassay42. Therefore, our study provides the first experimental data

to support the hypothesis that the conserved antagonist Arg-Phe-Phe residues may be

mimicking the agonist His-Phe-Arg-Trp interactions with melanocortin receptors,

specifically at the skin mMC1R.

Our finding that the decapeptide Tyr-[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr has

agonist activity at MC 1R is consistent with a model in which the conformational shape

that AGRP adopts determines its activity at its receptor. Since the decapeptide was

synthesized to mimic a single portion of the AGRP C-terminus, our study supports the

hypothesis that the amino acids adjacent to the decapeptide residues on the parent AGRP

peptide influence the conformational shape of the entire peptide. Thus, the spatial

relationships between the antagonist residues and the residues on the receptor are

important for determining the activity of AGRP at the melanocortin receptors.

Some experimental characteristics of our study resulted in some differences in our

findings and those from previous research in human melanocortin receptors and may

limit the comparison between our results and those previous. In particular, the binding

affinity that we found for the hAGRP at the mMC3R is 10-fold less potent than the

binding affinity previously reported for hMC3R. In addition, the binding affinity we

report for mMC4R is 4-fold less potent than the binding affinity reported for hMC4R.

We attribute these differences to two main experimental differences between our study

and previous studies: the species of receptor used and the differences in the radio-labeled

compound used. In our research, we used murine melanocortin receptors with the

intention of further reproducing our results in vivo in murine models, whereas previous

researchers have used human melanocortin receptors. Species differences have been









previously noted with melanocortin receptors42 and therefore further studies should be

conducted with human subjects to confirm our results. In addition, structural differences

distinguish the linear compound I125-NDP-MSH used by Tota et al. for binding activity

assays and the cyclic compound I125MTII that we used for these assays. However, despite

the differences in the radio-labeled compound used, we tested both I125-NDP-MSH and

I 25MTII at mMC R, mMC3R, mMC4R, and mMC5R and found them to have similar

ICso values at each receptor. Therefore, our binding assay results using I125MTII are

comparable to previous research with either compound.

Conclusion

Researchers have proposed that the Arg-Phe-Phe sequence located in the C-

terminus of agouti signal protein and AGRP is intimately involved in molecular

recognition and antagonist activity of these molecules and the melanocortin receptors.

The Arg-Phe-Phe sequence conserved in both of these compounds is structurally related

to the conserved sequence His-Phe-Arg-Trp found in the melanocortin-stimulating

hormone compounds. Furthermore, the Arg-Phe-Phe sequence may be mimicking the

interactions of the sequence His-Phe-Arg-Trp with the melanocortin receptors. The

results of this study provide evidence that supports this hypothesis.













APPENDIX
CHEMICAL STRUCTURES

Table A-i Chemical structures for FMOC amino acids


9-fluorenyl-methoxy-
carboxyl


(FMOC) Amino Acid


Fmoc-Arg(Pbf)


Fmoc-Asp(tBu)


Fmoc-Tyr (tBu)


Fmoc--NH OH


b











Table A-1 Continued


Reagent


Fmoc-Phe


Fmoc-Cys (Trt)


Fmoc-Ala


Fmoc-Tyr(tBu)-
Wang resin


Structure


OH

-NH-Fmoc


OH
Fmoa- NH
o










Table A-2 Chemical structures for FMOC reagents
Reagent Structure


benzotriazol-1-yl-oxy- N
tris(dimethylamino phosphonium
hexafluorophosphate \ F-F
(BOP) Inc .





1-hydroxybenzotriazole O
(HObt)




Table A-3 Various reagents for Solid Phase Synthesis
Benzotriazol-1 -yl-oxy-tris(dimethylamino)
phosphonium hexafluorophosphate 3-fold excess, 0.34g/amino acid
(BOP):
N,N-diisopropylethylamine (DIEA) 5.1-fold excess, 218 pL
10%: 50mL DIEA q.s. to 500mL with DMF
1-hydroxybenzotriazolemonohydrate (light sensitive): 27.2g HOBt q.s. to 200mL with DMF
(HOBt) (1M) (FW= 135.13) 760 uL (1 mmol)

N,N-dimethylformamide (DMF) (freshly distilled on sieves before use)


20% Piperidine in DMF (2:1)


Kaiser Solution A 0.01M potassium cyanate (KCN) (33mg KCN q.s to 50mL H2O)
added to 98mL Pyridine (bubbled with helium)

Kaiser Solution B
2.5g Ninhydrin q.s. to 50mL with n-butanol

Kaiser Solution C
80g phenol in 20mL n-butanol (leave in warm place overnight)
















REFERENCES


1. Flegal KM, Carroll MD, Ogden CL, Johnson CL. Prevalence and Trends in
Obesity Among US Adults, 1999-2000. JAMA. October 9, 2002
2002;288(14):1723-1727.

2. Flegal KM, Graubard BI, Williamson DF, Gail MH. Excess Deaths Associated
With Underweight, Overweight, and Obesity. JAMA. April 20, 2005
2005;293(15):1861-1867.

3. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD. Role of
melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature.
Jan 9 1997;385(6612):165-168.

4. Jackson IJ. Homologous pigmentation mutations in human, mouse and other
model organisms. Hum Mol Genet. 1997;6(10):1613-1624.

5. Cone RD. The central melanocortin system and its role in energy homeostasis.
Ann Endocrinol (Paris). Mar 1999;60(1):3-9.

6. Fisher SL, YagaloffKA, Bum P. Melanocortin-4 receptor: a novel signalling
pathway involved in body weight regulation. Int J Obes Relat Metab Disord. Feb
1999;23 Suppl 1:54-58.

7. Hadley ME, Haskell-Luevano C. The proopiomelanocortin system. Ann N YAcad
Sci. Oct 20 1999;885:1-21.

8. Solomon S. POMC-derived peptides and their biological action. Ann N YAcad
Sci. Oct 20 1999;885:22-40.

9. Starowicz K, Przewlocka B. The role of melanocortins and their receptors in
inflammatory processes, nerve regeneration and nociception. Life Sci. Jul 4
2003;73(7):823-847.

10. Fluck CE, Martens JWM, Conte FA, Miller WL. Clinical, Genetic, and
Functional Characterization of Adrenocorticotropin Receptor Mutations Using a
Novel Receptor Assay. J Clin EndocrinolMetab. September 1, 2002
2002;87(9):4318-4323.









11. Butler AA, Cone RD. Knockout Studies Defining Different Roles for
Melanocortin Receptors in Energy Homeostasis. Ann NYAcad Sci. June 1, 2003
2003;994(1):240-245.

12. Zhang L, Anthonavage M, Huang Q, Li W-H, Eisinger M. Proopiomelanocortin
Peptides and Sebogenesis. Ann NYAcad Sci. June 1, 2003 2003;994(1):154-161.

13. Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Walson SJ, DelValle J,
Yamada T. Molecular cloning, expression, and gene localization of a fourth
melanocortin receptor. J. Biol. Chem. July 15, 1993 1993;268(20):15174-15179.

14. Tritos NA, Maratos-Flier E. Two important systems in energy homeostasis:
melanocortins and melanin-concentrating hormone. Neuropeptides. Oct
1999;33(5):339-349.

15. Lu D, Willard D, Patel IR, Kadwell S, Overton L, Kost T, Luther M, Chen W,
Woychik RP, Wilison WO. Agouti protein is an antagonist of the melanocyte-
stimulating-hormone receptor. Nature. Oct 27 1994;371(6500):799-802.

16. Miller MW, Duhl DM, Vrieling H, Cordes SP, Ollman MM, Winkes BM, Barsh
GS. Cloning of the mouse agouti gene predicts a secreted protein ubiquitously
expressed in mice carrying the lethal yellow mutation. Genes Dev. Mar
1993;7(3):454-467.

17. Abdel-Malek Z, Suzuki I, Tada A, Im S, Akcali C. The Melanocortin-1 Receptor
and Human Pigmentation. Ann NYAcadSci. October 20, 1999 1999;885(1):117-
133.

18. Rana BK. New Insights into G-Protein-Coupled Receptor Signaling from the
Melanocortin Receptor System. Mol Pharmacol. July 1, 2003 2003;64(1):1-4.

19. Shutter JR, Graham M, Kinsey AC, Scully S, Luthy R, Stark KL. Hypothalamic
expression of ART, a novel gene related to agouti, is up- regulated in obese and
diabetic mutant mice. Genes andDevelopment. 1997; 11(5):593-602.

20. Millhauser GL, McNulty JC, Jackson PJ, Thompson DA, Barsh GS, Gantz I.
Loops and links: structural insights into the remarkable function of the agouti-
related protein. Ann N YAcad Sci. Jun 2003;994:27-35.

21. Bures EJ, Hui JO, Young Y, Chow DT, Katta V, Rohde MF, Zeni L, Rosenfeld
RD, Stark KL, Haniu M. Determination of disulfide structure in agouti-related
protein (AGRP) by stepwise reduction and alkylation. Biochemistry.
1998;37(35):12172-12177.









22. Ollmann MM, Wilson BD, Yang Y-K, Kerns JA, Chen Y, Gantz I, Barsh GS.
Antagonism of Central Melanocortin receptors in vitro and in vivo by agouti-
related protein. Science. 1997;278(5335):135-138.

23. Willard DH, Bodnar W, Harris C, Kiefer L, Nichols JS, Blanchard S, Hoffman C,
Moyer M, Burkhart W, Weiel J, Luther MA, Wilkison WO, Rocque WJ. Agouti
structure and function: Characterization of a potent [alpha]-melanocyte
stimulating hormone receptor antagonist. Biochemistry. 1995;34(38):12341-
12346.

24. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson
JD. Multiple sequence alignment with the Clustal series of programs. Nucleic
Acids Res. Jul 1 2003;31(13):3497-3500.

25. Tota MR, Smith TS, Mao C, MacNeil T, Mosley RT, Van der Ploeg LH, Fong
TM. Molecular interaction of Agouti protein and Agouti-related protein with
human melanocortin receptors. Biochemistry. Jan 19 1999;38(3):897-904.

26. Merrifield R. Solid-Phase Peptide Synthesis: 3. An Improved Synthesis of
Bradykinin. Biochemistry. September 1964 1964;14:1385-1390.

27. Stewart JM. Solidphasepeptide ,yimew\i\ San Francisco: W. H. Freeman; 1969.

28. Carpino L, Han G. The 9-fluorenylmethoxycarbonyl amino-protecting group.
Journal of Organic Chemistry. 1972;37:3404-3409.

29. Fields G. Solid-phase peptide syinhe\i\. San Diego: Academic Press; 1997.

30. Graham M, Shutter JR, Sarmiento U, Sarosi I, Stark KL. Overexpression of Agrt
leads to obesity in transgenic mice. Nat Genet. Nov 1997;17(3):273-274.

31. Stewart J, Young J. Solidphase peptide y/he/l/i\ Rockford, IL: Pierce Chemical;
1984.

32. Wang SS. p-alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide
resin for solid phase synthesis of protected peptide fragments. JAm Chem Soc.
Feb 21 1973;95(4):1328-1333.

33. Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection of free
terminal amino groups in the solid-phase synthesis of peptides. AnalBiochem.
Apr 1970;34(2):595-598.

34. Haskell-Luevano C, Shenderovich MD, Sharma SD, Nikiforovich GV, Hadley
ME, Hruby VJ. Design, synthesis, biology, and conformations ofbicyclic alpha-
melanotropin analogues. JMed Chem. May 12 1995;38(10):1736-1750.









35. Wigler M, Pellicer A, Silverstein S, Axel R, Urlaub G, Chasin L. DNA-mediated
transfer of the adenine phosphoribosyltransferase locus into mammalian cells.
Proc NatlAcad Sci USA. Mar 1979;76(3):1373-1376.

36. Jimenez A, Davies J. Expression of a transposable antibiotic resistance element in
Saccharomyces. Nature. Oct 30 1980;287(5785):869-871.

37. Al-Obeidi F, Castrucci AM, Hadley ME, Hruby VJ. Potent and prolonged acting
cyclic lactam analogues of alpha-melanotropin: design based on molecular
dynamics. JMed Chem. Dec 1989;32(12):2555-2561.

38. Chen W, Shields TS, Stork PJ, Cone RD. A colorimetric assay for measuring
activation of Gs- and Gq-coupled signaling pathways. Anal Biochem. Apr 10
1995;226(2):349-354.

39. Schild HO. pA, a new scale for the measurement of drug antagonism. 1947. Br J
Pharmacol. Feb 1997;120(4 Suppl):29-46; discussion 27-28.

40. Kiefer LL, Veal JM, Mountjoy KG, Wilkison WO. Melanocortin receptor binding
determinants in the agouti protein. Biochemistry. 1998;37(4):991-997.

41. Haskell-Luevano C, Hendrata S, North C, Sawyer TK, Hadley VJ, Dickinson C,
Gantz I. Discovery of prototype peptidomimetic agonists at the human
melanocortin receptors MC1R and MC4R. JMed Chem. Jul 4 1997;40(14):2133-
2139.

42. Mountjoy KG. The human melanocyte stimulating hormone receptor has evolved
to become "super-sensitive" to melanocortin peptides. Molecular and Cellular
Endocrinology. Jun 1994; 102(1-2):R7-R11.















BIOGRAPHICAL SKETCH

Anzeela Mulaiya Schentrup was born in Mississauga, Ontario, Canada, to parents

of Indian descent. She came to Florida at the age of 9 and grew up near Ft. Lauderdale.

She first attended the University of Florida in 1994 and pursued a Bachelor of Arts

degree in the history of science, in which she received highest honors for her thesis on

pharmacy in Germany at the onset of the Industrial Revolution. Through exposure to the

field of pharmacy sciences in the course of writing this thesis, Anzeela decided to pursue

a scientific career leading to a graduate degree in medicinal chemistry. After two years,

her scientific training stimulated her interest in a career in clinical medicine, so Anzeela

left medicinal chemistry to pursue a Doctor of Pharmacy degree, which she received in

2004. During this time, she was encouraged by her former mentor, Carrie Haskell-

Luevano, PhD, to pursue the Master of Science in medicinal chemistry for the work she

had already completed. In addition, after the birth of her second child, and due to the

faith of her current mentor, Julie Johnson, PharmD, Anzeela decided to return to graduate

research in pursuit of the Doctor of Philosophy degree in pharmacogenomics. While in

this degree program, Anzeela completed the Master of Science degree in medicinal

chemistry and continues to pursue her doctorate. Although her educational background is

diverse, all of the disciplines in which she has studied and developed a level of expertise

have contributed to her current understanding of pharmacy practice and ability to perform

translational research. Currently, Anzeela lives in Gainesville, Florida, with her husband,

Joseph C6te, and two children, Ena Marie and Maximilian C6te.