The melanocortin-4 receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating energy homeostasis and obesity. Up to a remarkable 6% of morbidly obese adults and children studied possess single nucleotide polymorphisms (SNPs) of the MC4R. Upon stimulation by agonist, the MC4R signals through the intracellular adenylate cyclase signal transduction pathway. Posttranslational modification of the pro-opiomelanocortin (POMC) gene transcript results in the generation of several endogenous melanocortin receptor agonists including alpha-, beta-, gamma-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH) ligands. The endogenous MC4R antagonist, agouti-related protein (AGRP), is expressed in the brain and is only one of two naturally occurring antagonists of GPCRs identified to date. Herein, we have generated 40 hMC4 polymorphic receptors and evaluated their cell surface expression by flow cytometry as well as pharmacologically characterized their functionality using the endogenous agonists alpha-MSH, beta-MSH, gamma2-MSH, ACTH(1-24), the antagonist hAGRP(87-132), and the synthetic agonists NDP-MSH and MTII. This is the first study in which polymorphic hMC4Rs have been pharmacologically characterized simultaneously with multiple endogenous ligands. Interestingly, at the N97D, L106P, and C271Y hMC4Rs beta-MSH was more potent than the other endogenous agonists alpha-MSH, gamma2-MSH, ACTH(1-24). The S58C and R165Q/W hMC4Rs possessed significantly reduced endogenous agonist potency (15- to 90-fold), but the synthetic ligands NDP-MSH and MTII possessed only 2-9-fold reduced potency as compared to the wild-type receptor, suggesting their potential as therapeutic ligands to treat individuals with these polymorphisms.

Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide template Tyr-c[Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that is equipotent to alpha-MSH at the mMC1, mMC3, and mMC5 receptors but is 30-fold more potent than alpha-MSH at the mMC4R. Additionally, these studies identified a new and novel >200-fold MC4R versus MC3R selective peptide Tyr-c[Asp-D-Phe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) template. Furthermore, when the His-DPhe-Arg-Trp sequence is used to replace the hAGRP Arg-Phe-Phe residues in the "mini"-AGRP (hAGRP87-120, C105A) template, a potent nanomolar agonist resulted at the mMC1R and MC3-5Rs.

Agouti-related protein (AGRP) is a potent orexigenic peptide that antagonizes the melanocortin-3 and -4 receptors (MC3R and MC4R). While the C-terminal domain of AGRP, AGRP(87-132), is equipotent to the full-length peptide, further truncation decreases potency at the MC3R and MC4R. Herein, we report AGRP-derived peptides designed to mimic the active β-hairpin secondary structure that contains the hypothesized Arg-Phe-Phe pharmacophore. The most potent scaffold, c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro], comprised the hexa-peptide β-hairpin loop from AGRP cyclized through a DPro-Pro motif. A 20 compound library was synthesized from this scaffold for further structure-activity relationship studies. The most potent peptide from this library was an Asn to diaminopropionic acid substitution that possessed sub-nM antagonist activity at the mMC4R and was greater than 160-fold selective for the mMC4R versus the mMC3R. The reported ligands may serve as probes to characterize the melanocortin receptors in vivo and leads in the development of novel therapeutics.

The melanocortin system has been implicated in a multitude of physiological pathways including obesity, satiety, energy homeostasis, sexual behavior, pigmentation, sodium regulation, hypertension, and many others. Based upon studies of the endogenous melanocortin receptor agonists at the cloned human melanocortin receptor proteins, it was concluded that the γ-MSH related agonist ligands are selective for the MC3 versus the MC4 and MC5 receptors. In attempts to understand and identify the specific amino acids of γ 2 -MSH important for MC3R selectivity, we have performed Nand C-terminal truncation studies and pharmacologically characterized twenty-eight ligands at the mouse MC1 and MC3-5 melanocortin receptors. The C-terminal Trp-Asp 9 -Arg 10 -Phe 11 residues are important for nM potency at the mMC3R and the Arg 7 -Trp 8 residues are important for mMC5R nM potency. We observed the unanticipated results that several of the C-terminal truncated analogues possessed nM agonist potency at the mMC3 and mMC5Rs which lead us to performed a comparative side-by-side study of the mouse and human MC5R. These data resulted in μM γ 2 -MSH analogue potency at the hMC5R, consistent with previous reports, however at the mMC5R, nM γ 2 -MSH analogue potency was observed. Thus, these data support the hypothesis of important species specific differences in γ-MSH related ligand potency at the rodent versus human MC5R subtype that is critical for the interpretation of in vivo rodent physiological studies. These results prompted us to examine the affects of a peripherally administered melanocortin agonist on hypothalamic gene expression levels of the MC3R, MC4R, and MC5R. The super potent non-selective NDP-MSH agonist was administered i.p. and resulted in significantly decreased levels of mMC3R and mMC5R hypothalamic mRNA versus saline control. These data provide for the first time data demonstrating peripherally administered NDP-MSH can modify hypothalamic melanocortin receptor expression levels. KeywordsMelanotropin; heart; blood pressure; obesity; receptor brain expression; GPCR © 2010 Elsevier Inc. All rights reserved. * Reprint request should be addressed to Dr. Carrie Haskell-Luevano, University of Florida, Department of Pharmacodynamics, P. O. Box 100487, Gainesville, FL 32610-0487. Phone (352) Fax (352) 273-7723, carrie@cop.ufl.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptPeptides. Author manuscript; available in PMC 2011 December 1. Published in final edited form as:Peptides. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA...

The melanocortin system has been implicated in regulating various physiological processes including pigmentation, energy homeostasis, obesity, steroidogenesis cardiovascular, and exocrine gland function. The five melanocortin receptors that belong to the super family of G protein-coupled receptors are stimulated by naturally occurring agonists. The aim of this research was focused on the design, synthesis, and pharmacological characterization of melanocortin ligands that contain the 1,2,5-trisubstituted benzimidazole scaffold. A series of benzimidazole analogues, with three points of diversity at positions 1, 2, and 5, were designed, synthesized, pharmacologically assayed at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R and resulted in ligands possessing a range of agonist activity from nm to no stimulation at up to 100 microM concentrations. This study demonstrates that the benzimidazole structure template can be appended with key melanocortin agonist amino acids for the design melanocortin receptor agonist ligands.