By use of a solid-phase synthetic approach, a bioactive reverse turn heterocycle was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R−MC5R) are G-protein-coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nanomolar potency. Biophysical structural analysis [2D 1H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families compared to the reference peptide and result in a novel template for further structure−function studies.

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 feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [α-, β-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87−132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH2 (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R’s and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87−132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

The melanocortin pathway has emerged during this past decade as an important target area for the discovery and development of therapeutic agents related to obesity and type 2 diabetes. This peptide-G-protein coupled receptor (GPCR) pathway has evolved from peptide-based ligands to small molecules possessing a variety of different molecular scaffolds. Herein, we summarize the originating hypothesis of the importance of the reverse β-turn secondary structure for agonist ligand potency at the melanocortin receptors and how that information was utilized for the discovery of small molecules based upon this type of turn structure.

The melanocortin system consists of five seven-transmembrane spanning G-protein coupled receptors (MC1−5) that are stimulated by endogenous agonists and antagonized by the only two known endogenous antagonists of GPCRs, agouti and agouti-related protein (AGRP). These receptors have been associated with many physiological functions, including the involvement of the MC4R in feeding behavior and energy homeostasis, making this system an attractive target for the treatment of obesity. Small-molecule mimetics of endogenous ligands may result in the development of compounds with properties more suitable for use as therapeutic agents. The research presented herein involves the synthesis and analysis of 12 melanocortin receptor agonists using the 1,4-benzodiazepine-2,5-dione template and is the first report of these derivatives as melanocortin receptor agonists. Structure–activity relationship studies using this privileged structure template has resulted in molecules with molecular weights around 400 that possess nanomolar agonist potency at the melanocortin receptors examined in this study.

The melanocortin-3 and -4 receptors (MC3R, MC4R) have been implicated in energy homeostasis and obesity. Whereas the physiological role of the MC4R is extensively studied, little is known about the MC3R. One caveat is the limited availability of ligands that are selective for the MC3R. Previous studies identified Ac-His-DPhe(p-I)-Arg-Trp-NH2, which possessed partial agonist/antagonist pharmacology at the mMC3R while retaining full nanomolar agonist pharmacology at the mMC4R. These data allowed for the hypothesis that the DPhe position in melanocortin tetrapeptides can be used to examine ligand side-chain determinants important for differentiation of mMC3R agonist versus antagonist activity. A series of 15 DPhe7 modified Ac-His-DPhe7-Arg-Trp-NH2 tetrapeptides has been synthesized and pharmacologically characterized. Most notable results include the identification of modifications that resulted in potent antagonists/partial agonists at the mMC3R and full, potent agonists at the mMC4R. These SAR studies provide experimental evidence that the molecular mechanism of antagonism at the mMC3R differentiates this subtype from the mMC4R.

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 γ2-MSH important for MC3R selectivity, we have performed N- and C-terminal truncation studies and pharmacologically characterized twenty-eight ligands at the mouse MC1 and MC3-5 melanocortin receptors. The C-terminal Trp-Asp9-Arg10-Phe11 residues are important for nM potency at the mMC3R and the Arg7-Trp8 residues are important for mMC5R nM potency. We observed the unanticipated results that several of the C-terminal truncated analogs possessed nM agonist potency at the mMC3 and mMC5Rs which lead us to perform a comparative side-by-side study of the mouse and human MC5R. These data resulted in μM γ2-MSH analog potency at the hMC5R, consistent with previous reports, however at the mMC5R, nM γ2-MSH analog 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.Graphical abstractDownload full-size imageResearch highlights▶ Identification of γ2-MSH peptide amino acids important for melanocortin receptor potency. ▶ The γ2-MSH ligands have different mouse versus human species specific potencies. ▶ Intraperitoneal (i.p.) injection of NDP-MSH into mice reduces hypothalamic MC3R and MC5R mRNA gene expression levels.