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Universit y of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 1 Amino Acids for the Melanocortin System Viktor Flores Dr. Anamika Singh Huisuo Huang, and Dr. Carrie Haskell Luevano College of Medicine University of Florida The melanocortin system consists of five G protein coupled r eceptors (MC1R MC5R), four known endogenous agonists, and two known endogenous antagonists. The MC3R and MC4R play a direct role in regulating energy and weight homeostasis. When the MC3R and MC4R are stimulated by their agonist ligands, an anorexigenic re sponse is produced. All four of the endogenous agonist ligands of the melanocortin system have the core tetrapeptide sequence His Phe Arg Trp which has been attributed to melanocortin receptor stimulation. The purpose of this study is to modify each amino acid of the enhanced tetrapeptide His DPhe Arg Trp with its amino acid counterpart and test the selectivity and potency that each change will produce at the MC3R and MC4R. The peptides were synthesized using standard Fmoc solid phase peptide methodolog y. The synthesized peptides were pharmacologically galactosidase gene reporter assay. Compared to the conserved tetrapeptide sequence found in endogenous agonists, the Arg Trp analogue were 400 and 1000 fold more potent at th e MC4R. T he and analogues are potent agonist selective for the MC4R and may be useful for studying MCRs in order to design a drug that regulates weight homeostasis and food satiety. INTRODUCTION Melanocortin System The melanocor tin system is a complex control center that regulates various physiological pathways, which include skin pigmentation, steroidogenesis, sexual function, sebaceous lipid production, energy and weight homeostasis, and food satiety [1,2] This system consis ts of five G protein coupled receptors (MC1R MC5R), four known endogenous agonists, and two known endogenous antagonists [2, 3] The endogenous agonists of the melanocyte MSH) and adrenocorticotropin hormone (ACTH). All four of these endogenous agonist ligands are derived from the posttranslational processing of the proopicomelanocortin (POMC) gene [4,5] These hormones bind to the melanocortin receptors and activate the cyclic adenosine monophosphate (cAMP) signal transduction pathway, which generates a cascade of intracellular reactions that result in a physiolo gical response  The endogenous antagonists are Agouti and Agouti related protein AgRP [7, 8] These proteins inhibit the function of the agonists by [6 8] Of the five melanocortin receptors, the MC3R and MC4R have generated the greatest interest among the scientific community because of their direct role in regulating energy and weight homeostasis [9, 10] In MC3R studies, mice null for MC3R developed higher levels of fat mass and lower level s of muscle mass despite eating a similar diet to the control mice, establishing a connection between the MC3R and energy homeostasis [11,12] MC4R studies have revealed even more dramatic results. Knockout MC4R mice have been shown to be hyperphagic and o vertly obese, proving that this receptor is critical for weight homeostasis and feeding behavior [13 17] In addition, clinical studies have shown that people with MC4R mutations are usually morbidly obese [16, 17] In fact, these studies suggest that muta tions in the gene encoding the MC4R account for the most common form of monogenic obesity seen in humans. Up to 6% of morbidly obese individuals express a mutation in the MC4R  Studies have also determined the relationship between these receptors and their endogenous agonists and antagonists. A study by Fan et al. concluded that food intake in mice is suppressed by agonists and activated by antagonists when either agent is administered intracerebroventricularly near the hypothalamus of the brain  Following all these important findings, scientists have successfully constructed the molecular pathways that account for these observations. Our current understanding of how the MC3R and MC4R regulate energy and weight homeostasis involves the interaction between peripheral energy stores and the arcuate nucleus of the hypothalamus  The body naturally balances caloric input with caloric expenditure by storing excess energy in adipose tissue, a major peripheral storage site. An increase of adipose tissue results in an increase of leptin hormone expression. The leptin hormone is released into the bloodstream and travels to the brain, where it binds to leptin receptors expressed in POMC neurons inside the arcuate nucleus of the hypothalamus [19, 20] The POM C MSH,
VIKTOR FLORES D R A NAMIKA S INGH H UISUO H UANG AND D R C ARRIE H ASKELL L UEVANO University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 2 which mediates an anorexigenic response when it binds to the MC3R and MC4R [5, 20] The converse patterns of events are also true; when the body is in a fasting state, POMC gene expression decreases and AgRP expression increases in AgRP/NPY neurons [21, 22] Subsequently, MSH stimulation, which results in an orexigenic response [6 8] anorexigenic response by reducing food intake, efforts to develop a therapeutic drug against obesity have aimed to MSH and ACTH all share the same tet rapeptide pharmacore of His Phe Arg Trp (HFRW) [23 25] This is the minimal sequence required for melanocortin receptor selectivity and stimulation  Figure 1 compares the sequence of each endogenous agonist. Moreover, it was found that the stereochemi cal inversion of Phe into D Phe results in a more potent and enzymatically stable tetrapeptide that targets the human MC4R  Figure 1. The endogenous agonists of the melanocortin s ystem This is the single letter amino acid abbreviation for the four known endogenous agonists of the melanocortin system. Encircled is the core tet rapeptide sequence of histidine phenylalanine arginine t ryptophan, which is necessary for melanocortin receptor stimulation. Amino Acids The purpose of this study is to modify each amino acid of the His DPhe Arg amino acid counterpart and test the selectivity and potency that each change will produce on MC3R and MC4R. Unlike their amino acids dif fer in that they have an extra carbon between 3 amino 2 amino acids)  3 amino acids. amin 3 amino acid. Figure 2. 3 amino acid amino acid has an extra c arbon i n the backbone of its structure amino acids for drug development and peptidomimetics are promising because of three reasons: peptides, can have multiple conformations and well define d secondary structures due to their extended backbone [28,29] peptides can be designed to resemble the activity related structural features of their corresponding natural peptides can be designed to biological function [30 33] In fact, peptides can mimic the effector response of antibodies, anticancer proteins, anti HIV molecules, and vaccines [30 33] peptides are enzymatically stable against proteolytic, hydrolytic, and metabolic enzymes in mammals because these enzymes do not recognize the additional carbon in the backbone of the amino acid [29,34] peptides would be expected to last longer in vivo peptides, a factor that is very important when consi dering d rug development. METHODS Materials amino acids ( S ) 3 Fluorenylmethoxycarbonyl 4 phenylbutyric acid L homophenylalanine (Fmoc HoPhe OH), Fluorenylmethoxycarbonyl N w (2,2,5,7,8 penta methyl chromane 6 sulfonyl) L homoarginine (Fmoc HoA rg(Pmc) OH), and ( S ) 3 Fluorenylmethoxycarbonyl 4 (3 indolyl) butyric acid L homotryptophan (Fmoc HoTrp OH) were purchased from Chem Impex International (Wood Dale, IL). The amino acids N 9 Fluorenylmethoxycarbonyl Nim Trityl L Histidine (Fmoc His(Tr t) OH), 9 Fluorenylmethoxycarbonyl L Phenylalanine ( Fmoc Phe OH), 9 Fluorenylmethoxycarbonyl D Phenylalanine
Universit y of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 3 (Fmoc D Phe OH), and N 9 Fluorenylmethoxycarbonyl N in t Butyloxycarbonyl L Tryptophan (Fmoc Trp(Boc) OH) were purchased from Peptide Internatio nal (Louisville, KY). The solid support resin 4 (2',4' Dimethoxyphenyl Fmoc aminomethyl) phenoxyacetamido norleucyl Methylbenzhydrylamine resin (Rink Amide MBHA; 0.37 mequiv/g), and the coupling reagent 1 [bis(Dimethylamino)methylene] 1H Benzotriazolium He xafluorophosphate 3 Oxide (HBTU) were also purchased from Peptides International. N N Diisopropylethylamine (DIEA) and triisopropylsilane (TIS) were purchased from Aldrich (Milwaukee, WI). Acetonitrile (ACN), anhydrous ethyl ether, dichloromethane (DCM), g lacial acetic acid (HOAc) and methanol (MeOH) were purchased from Fisher (Fair Lawn, NJ). N,N dimethylformamide (DMF) was purchased from Burdick and Jackson (McGaw Park, IL). Trifluoroacetic acid, piperidine, and pyridine were purchased from Sigma (St. Lou is, MO). 1,2 Ethanedithiol was purchased from Fluka. Peptide Synthesis Peptide synthesis was performed using standard Fmoc methodology on a CEM Discovery SPS microwave peptide synthesizer  Approximately 270mg of Rink Amide MBHA was placed inside a rea ction vessel and allowed to swell for 2 hours in dichloromethane (DCM) prior to use. The resin was washed five times with DMF and deprotected using 20% piperidine in DMF solution for 2 minutes under normal room temperature followed by another 20% piperidin e in DMF treatment but this time inside the microwave synthesizer for 4 minutes at 75 C. To confirm the deprotection of the amine group, a Kaiser test was performed  The growing peptide chain was synthesized on the amide resin using the following c oupling steps: Three fold excess of the appropriate Fmoc amino acid, starting from the C terminus of the desired peptide, was added to the reaction vessel followed by the addition of three fold excess of HBTU and DIEA. The reaction vessel was then placed i n the microwave synthesizer at a setting of 75C for 5 minutes under N 2 bubbling. After 5 minutes, the reaction was allowed to cool down, the excess reagents were drained out, and the nascent peptide was washed with DMF four times. A Kaiser test was perfor med again to make sure the amino acid coupled with the resin. Following a successful coupling, the peptide was deprotected using 20% piperidine in DMF for 2 minute and then again for 4 minutes at 75C using the microwave synthesizer. Coupling and deprotect ion of the peptide was repeated until the final amino acid was added. Following the deprotection of the final amino acid, the peptide was acetylated by adding 3:1 mixture of glacial acetic acid and pyridine to the vessel for 30 minutes under N 2 bubbling. T his step was performed to protect the exposed N terminus. The solution was drained out, the peptide was washed with DCM four times, and the vessel was placed inside a vacuum overnight. Cleavage of the peptide from the resin and removal of the side chain pr otecting groups from the amino acids was done by adding a cleavage cocktail consisting of 91% TFA, 3% EDT, 3% TIS, and 3% H 2 O in the vessel for 3 hours under N 2 bubbling The cleavage product was emptied into a preweighted 50mL conical tube and precipitate d with cold anhydrous ethyl ether. The flocculent peptide was pelleted by centrifugation ( Sorval Super T21 high speed centrifuge ) at 4C and 4000rpm for 4 minutes. The anhydrous ethyl ether was decanted from the centrifuge tube and the peptide was again mi xed with cold anhydrous ethyl ether and centrifuged under the same setting. This step was repeated two more times. The crude peptide inside the conical tube was placed inside a vacuum overnight. Peptide Purification Approximately 20mg of the dry crude pept ide was purified by reverse phase high liquid performance chromatography (RP HPLC) using a Shimadzu chromatography system with a photodiode array detector and a semipreparative RP HPLC C 18 bonded silica column. The major peak was collected and the solvents used to purify the crude peptide (acetonitrile, methanol, and water) were removed by rotovaporization and lyophilization. The In order to verify that the final product was the desired peptide, mass spectrometry was performed. The mass spectrometry results were compared to the calculated molecular mass of each peptide (University of Florida protein core facility). Galactosidase Ass ay galactosidase assays were performed in the Haskell Luevano laboratory Pharmacological character ization of the synthesized peptides at the mouse melanocortin receptors mMC1R, mMC3R, mMC4R, and galactosidase gene reporter assay as described by Chen et al.  The mMC2R was not utilized for characte rization because this receptor only selects for ACTH agonist  RESULTS Purification of Peptides Table 1 reports the analytical data of the synthesized the purified peptides under the solvent acetonitrile and methanol. This value is important for reproducibility purposes. The purified peptides were at least 95% pure as determined by the analytical RP HPLC. The mass spectrometry results confirmed that the correct peptides were synthesized.
VIKTOR FLORES D R A NAMIKA S INGH H UISUO H UANG AND D R C ARRIE H ASKELL L UEVANO University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 4 Table 1. Analytical Data for the Peptides Synthesized Peptide Acetonitrile HPLC Methanol HPLC Purity % m/Z Calculated m/Z Found Ac His Phe Arg Trp NH2 4.6 6.8 >95 685.34 686.56 Ac His DPhe Arg Trp NH2 4.0 6.0 >95 685.34 686.62 Ac His Arg Trp NH2 4.4 6.6 ~ 95 699.36 700.57 Ac His DPhe Trp NH2 3.9 6.0 >95 699.36 700.26 Ac His DPhe Arg NH2 4.1 6.3 >97 699.36 700.44 Note. es: Acetonitrile and Methanol. This value was determined using Equation 1. The calculated mass over charge values represent the expected mass of the peptides and the found mass over charge values is the actual mass of the peptides determined by mass spectrometry. Evaluation of Synthes ized Peptides at Melanocortin Receptors Table 2 summarizes the agonist pharmacology of each synthesized peptide at the mouse MC1R, MC3R, MC4R, and MC5R. NDP MSH, and it is used as a molecular probe for studying melanocortin receptors and comparing the potency of synthesized agonists  Peptide 1 is the control tetrapeptide sequence common to all four endogenous agonists, HFRW. Stimulation from NDP MSH resulted in 940000 162000 126000 and 32000 fold increased potencies at MC1R, MC3R, MC4R, and MC5R respectively, relative to Peptide 1 Peptide 2 which has DPhe instead of Phe, resulted in 3200 550 2020 and 1260 fold in creased potencies at the MC1R, MC3R, MC4R, and MC5R relative to Peptide 1 Peptide 3 which Phe, has increased potencies of 5 3 and 2 fold at the MC1R, MC4R, and MC5R respectively, relative to 1 Peptide 3 activity at the MC3R is equipotent to Peptide 1 Peptide 4 is similar to 2 with the only difference being that Arg instead of Arg. Potencies for 4 relative to Peptide 2 resulted in 12 5 and 12 fold decrease at the MC3R, MC4R, and MC5R respectively. Peptide 4 resulted in equipotent activity at the MC1R compared to 2. Peptide 5 is similar to 2 but cont Trp rather than Trp. Peptide 5 resulted in potencies of 2 9 and 2 fold decrease at the MC1R, MC3R, and MC4R respectively, relative to Peptide 2 At the MC5R, Peptide 2 and 5 had equipotent activity. Figures 3 through 6 illustrate the molar agoni st EC 50 values of each peptide at the mMC1R, mMC3R, mMC4R, and mMC5R. Table 2. Biological Activity of Tetrapeptide Agonists at the Mouse Melanocortin Receptors Peptide Peptide Structure Agonist EC50 (nM) Values mMC1R mMC3R mMC4R mMC5R NDP MSH 0.0 30.014 0.200.021 0.050.002 0.170.05 1 Ac His Phe Arg Trp NH2 281007480 3230014100 6310623 5490788 2 Ac His DPhe Arg Trp NH2 8.762.45 58.49.22 3.130.29 4.351.47 3 Ac His Arg Trp NH2 54601300 257009200 2320389 2710550 4 Ac His DPhe Trp NH2 11.01.41 711148 15.90.34 52.922.3 5 Ac His DPhe Arg NH2 14.02.72 503144 6.311.21 6.491.94 Note. The EC 50 values represent the standard error of mean as determined from three individual experiments.
PEPTIDE ANALOGUES CO AMINO A C IDS FOR THE M ELA NOCORTIN S YSTEM University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 5 Figure 3 Tetrapeptide mMC1R s timulatory a ctivity Stimulatory agonist activity of each synthesized peptide at the mMC1R. The EC 50 value for each analogue was 0.03nM (NDP M 5). Figure 4 Tetrapeptide mMC3R Stimulatory Activity Stimulatory agonist activity of each synthesized peptide at the mMC3R. The EC 50 value for each analogue was 0.20nM (NDP MSH 5). Figure 5 Tetrapeptide mMC4R Stimulatory Activity Stimulatory agonist activity of each synthesized peptide at the mMC4R. The EC 50 value for each analogue was 0.05nM (NDP (Peptide 5). Figure 6 Tetrapeptide mMC5R Stimulato ry Activity. Stimulatory agonist activity of each synthesized peptide at the m MC5R. The EC 50 value for each analogue was 0.17nM (NDP (Peptide 5). DISCUSSION via action on five G p rotein coupled receptor subtypes, MC1R MC5R. This is the first study to test the amino acids have at the melanocortin receptors. However, this is not the first study amino acid activity in G protein coupled receptor s. Coincidentally, a study in 2003 by Nunn et al. tetrapeptide analogues of the hormone somatostatin behave as potent agonists at somatostatin sst 4 receptor one of five G protein coupled receptors that make up the somatostatin system, also located in the brain  amino acids can also interact with G protein coupled receptors of the melanocortin system. amino acid of the tetrapeptide His DPhe Arg Trp (with the exception of histidine) was amino acid counterpart, and the potency and selectivity of each analogue was measured at the mouse melanocortin His so that we can measure the potency and selectivity of Ac DPhe Arg Trp NH 2 and Ac NH 2 at the mMCRs. amino Acid Containing Peptides amino acid tetrapeptides (Ac His Arg Trp NH 2 Ac His DPhe Trp NH 2, and Ac His DPhe Arg NH 2 ; summarized in table 2) show that each peptide was capable of stimulating the exp ressed melanocortin receptors. The most significant results of this study include the potency and selectivity displayed by Ac His DPhe Trp NH 2 and Ac His DPhe Arg NH 2 Ar g analogue
VIKTOR FLORES D R A NAMIKA S INGH H UISUO H UANG AND D R C ARRIE H ASKELL L UEVANO University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 6 was selective for MC1R and MC4R. Compared to the conserved tetrapeptide sequence found in endogenous agonists, Ac His Phe Arg Trp NH 2 Arg analogue was 400 fold more potent at MC4R, the receptor that pharmacologist are most interested in t argeting for obesity. Arg was only 5 fold less potent at the MC4R than the enhanced tetrapeptide, Ac His DPhe Arg Trp NH 2. Trp analogue was selective for MC1R, MC4R, and MC5R; this peptide was 1000 fold more potent than Ac His Phe Arg Trp NH 2 and only 2 fold less potent than the enhanced tetrapeptide, Ac His DPhe Arg Trp NH 2 Trp analogues were less potent than the already established Ac His DPhe Arg Trp NH 2 nd that these two peptides may still be useful for drug design amino acid containing peptides are more amino acid containing amino acid tetrapeptides showed selectivity for MC3R. Phe Position Another interesting result derived from this study is the importance of the Phe position for ligand efficacy. Previous studies found that stereochemically modifying His Phe Arg Trp to His DPhe Arg Trp, dramatically increased potency [25 ,26] In our study, changing Phe to DPhe resulted in an increase potency of 2000 fold at the MC4R. Thus, this study not only confirmed the results of previous findings, but als o fold decreased potency relative to Ac His DPhe Arg Trp NH 2 CONCLUSION In conclusion, the Ac His DPhe Trp NH 2 and Ac His DPhe Arg NH 2 peptides, with ligand efficacy of 15.9nM and 6.31nM respectively, are potent agonist selective for MC4R. In a ddition, the potential resistance of enzymatic degradation offered by these two analogues makes them useful ligands for studying MCRs and designing a drug that can regulate weight homeostasis and food satiety. Future Direction His so that the selectivity and potency of Ac DPhe Arg Trp NH 2 and Ac NH 2 at melanocortin receptors can be characterized. All the synthesized peptides must then be tested for in vitro enzymatic resistivity to determine whether the amino acids actually increases enzymatic stability. ACKNOWLEDGEMENT S I thank Dr. Carrie Haskell Luevano for the opportunity to work in her lab. None of this would have been possible without her. I also thank Dr. Anamika Singh f or her ment orship and patience and my colleagues at the Haskell Luevano lab for their support and help. REFERENCES 1. Haskell Luevano, Carrie, et al. "Progress in the Development of Melanocortin Receptor Selective Ligands." Current Pharmaceutical Design 10 (2004): 3443 3479. 2. Gantz, Ira, and Tung M. Fung. "The Melanocortin System." American Journal of Physiology of Endocrinology and Metabolism 284 (2003): E468 474. 3. Holder, J. R., and C. Haskell Luevano. "Melanocortin Ligands: 30 Years of Structure Activity Relat ionship (SAR) Studies." Medicinal Research R eviews 24.3 (2004): 325 56. 4. Gee, C. E., et al. "Identification of Proopiomelanocortin Neurones in Rat Hypothalamus by in Situ cDNA mRNA Hybridization." Nature 306.5941 (1983): 374 6. 5. Pritchard, L. E., A. V. T urnbull, and A. White. "Pro Opiomelanocortin Processing in the Hypothalamus: Impact on Melanocortin Signalling and Obesity." The Journal of E ndocrinology 172.3 (2002): 411 21. 6. Cone, R. D., et al. "The Melanocortin Receptors: Agonists, Antagonists, and th e Hormonal Control of Pigmentation." Recent Progress in Hormone R esearch 51 (1996): 287,317; discussion 318. 7. Lu, D., et al. "Agouti Protein is an Antagonist of the Melanocyte Stimulating Hormone Receptor." Nature 371.6500 (1994): 799 802. 8. Ollmann, M. M. et al. "Antagonism of Central Melanocortin Receptors in Vitro and in Vivo by Agouti Related Protein." Science 278.5335 (1997): 135 8. 9. Seeley, Randy J., Deborah L. Drazen, and Deborah J. Clegg. "The Critical Role of the Melanocortin System in the Contro l of Energy Balance." Annual Review of Nutrition 24.1 (2004): 133 49. 10. O'Rahilly, S., G. S. Yeo, and I. S. Farooqi. "Melanocortin Receptors Weigh in." Nature M edicine 10.4 (2004): 351 2. 11. Chen, A. S., et al. "Inactivation of the Mouse Melanocortin 3 Rece ptor Results in Increased Fat Mass and Reduced Lean Body Mass." Nature Ge netics 26.1 (2000): 97 102. 12. Butler, A. A., et al. "A Unique Metabolic Syndrome Causes Obesity in the Melanocortin 3 Receptor Deficient Mouse." Endocrinology 141.9 (2000): 3518 21. 13. Yeo, G. S., et al. "A Frameshift Mutation in MC4R Associated with Dominantly Inherited Human Obesity." Nature G enetics 20.2 (1998): 111 2. 14. Tao, Y. X., and D. L. Segaloff. "Functional Characterization of Melanocortin 4 Receptor Mutations Associated with Childhood Obesity." Endocrinology 144.10 (2003): 4544 51. 15. Huszar, D., et al. "Targeted Disruption of the Melanocortin 4 Receptor Results in Obesity in Mice." Cell 88.1 (1997): 131 41.
PEPTIDE ANALOGUES CO AMINO A C IDS FOR THE M ELA NOCORTIN S YSTEM University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 20 12 7 16. Vaisse, C., et al. "Melanocortin 4 Receptor Mutations are a Frequent and Heterogeneous Cause of Morbid Obesity." The Journal of Clinical I nvestigation 106.2 (2000): 253 62. 17. Farooqi, I. S., et al. "Clinical Spectrum of Obesity and Mutations in the Melanocortin 4 Receptor Gene." The New England Journal of M edicine 348.12 ( 2003): 1085 95. 18. Fan, W., et al. "Role of Melanocortinergic Neurons in Feeding and the Agouti Obesity Syndrome." Nature 385.6612 (1997): 165 8. 19. Schwartz, M. W., et al. "Identification of Targets of Leptin Action in Rat Hypothalamus." The Journal of Clin ical I nvestigation 98.5 (1996): 1101 6. 20. Cowley, M. A., et al. "Leptin Activates Anorexigenic POMC Neurons through a Neural Network in the Arcuate Nucleus." Nature 411.6836 (2001): 480 4. 21. Mizuno, T. M., and C. V. Mobbs. "Hypothalamic Agouti Related Prot ein Messenger Ribonucleic Acid is Inhibited by Leptin and Stimulated by Fasting." Endocrinology 140.2 (1999): 814 7. 22. Cowley, M. A., et al. "Integration of NPY, AGRP, and Melanocortin Signals in the Hypothalamic Paraventricular Nucleus: Evidence of a Cell ular Basis for the Adipostat." Neuron 24.1 (1999): 155 63. 23. Hruby, V. J., et al. "Alpha Melanotropin: The Minimal Active Sequence in the Frog Skin Bioassay." Journal of Medicinal C hemistry 30.11 (1987): 2126 30. 24. Castrucci, A. M., et al. "Alpha Melanotro pin: The Minimal Active Sequence in the Lizard Skin Bioassay." General and Comparative E ndocrinology 73.1 (1989): 157 63. 25. Haskell Luevano, C., et al. "Characterization of Melanocortin NDP MSH Agonist Peptide Fragments at the Mouse Central and Peripheral Melanocortin Receptors." Journal of Medicinal Ch emistry 44.13 (2001): 2247 52. 26. Haskell Luevano, C., et al. "Discovery of Prototype Peptidomimetic Agonists at the Human Melanocortin Receptors MC1R and MC4R." Journal of Medicinal C hemistry 40.14 (1997): 21 33 9. 27. Chemical Communications 21 (1997): 2015 22. 28. Beke, Tams, et al. peptide Structures." Journal of Computational Chemistry 27.1 (2006): 20 38. 29. Wu, Y. D., et al. "Theoretical Analysis of Secondary Structures of Beta Peptides." Accounts of Chemical Research 41.10 (2008): 1418 27. 30. Webb, A. I., et al. "T Cell Determinants Incorporating Beta Amino Acid Residues are Protease Resistant and Remain Immunogenic in Viv o." Journal of I mmunology (Baltimore, Md.: 1950) 175.6 (2005): 3810 8. 31. Kritzer, J. A., et al. "Helical Beta Peptide Inhibitors of the p53 hDM2 Interaction." Journal of the American Chemical Society 126.31 (2004): 9468 9. 32. Porter, E. A., et al. "Non Haem olytic Beta Amino Acid Oligomers." Nature 404.6778 (2000): 565. 33. Stephens, O. M., et al. "Inhibiting HIV Fusion with a Beta Peptide Foldamer." Journal of the American Chemical Society 127.38 (2005): 13126 7. 34. Disney, M. D., et al. "N Linked Glycosylated B eta Peptides are Resistant to Degradation by Glycoamidase A." Chemistry and B iodiversity 2.12 (2005): 1624 34. 35. Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis; 2 nd ed.; Pierce Chemical Co.: Rockford, IL, 1984. 36. Kaiser, E., et al. "Color Test f or Detection of Free Terminal Amino Groups in the Solid Phase Synthesis of Peptides." Analytical Biochemistry 34.2 (1970): 595 8. 37. Chen, W., et al. "A Colorimetric Assay for Measuring Activation of Gs and Gq Coupled Signaling Pathways." Analytical Bioche mistry 226.2 (1995): 349 54. 38. Schioth, H. B., et al. "Major Pharmacological Distinction of the ACTH Receptor from Other Melanocortin Receptors." Life Sciences 59.10 (1996): 797 801. 39. Sawyer, T. K., et al. "4 Norleucine, 7 D Phenylalanine Alpha Melanocyte Stimulating Hormone: A Highly Potent Alpha Melanotropin with Ultralong Biological Activity." Proceedings of the National Academy of Sciences of the United States of America 77.10 (1980): 5754 8. 40. Nunn, C., et al. "Beta(2)/beta(3) Di and alpha/beta(3) Te trapeptide Derivatives as Potent Agonists at Somatostatin Sst(4) Receptors." Naunyn Schmiedebe rg's Archives of P harmacology 367.2 (2003): 95 103.
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