The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

The discovery of the endogenous melanocortin agonists in the 1950s have resulted in sixty years of melanocortin ligand research. Early efforts involved truncations or select modifications of the naturally occurring agonists leading to the development of many potent and selective ligands. With the identification and cloning of the five known melanocortin receptors, many ligands were improved upon through bench-top in vitro assays. Optimization of select properties resulted in ligands adopted as clinical candidates. A summary of every melanocortin ligand is outside the scope of this review. Instead, this review will focus on the following topics: classic melanocortin ligands, selective ligands, small molecule (non-peptide) ligands, ligands with sex-specific effects, bivalent and multivalent ligands, and ligands advanced to clinical trials. Each topic area will be summarized with current references to update the melanocortin field on recent progress.

The melanocortin system has five receptors, and antagonists of the central melanocortin receptors (MC3R, MC4R) are postulated to be viable therapeutics for disorders of negative energy balance such as anorexia, cachexia, and failure to thrive. Agouti-related protein (AGRP) is an antagonist of the MC3R and an antagonist/inverse agonist of the MC4R. Biophysical NMR-based structural studies have demonstrated that the active sequence of this hormone, Arg-Phe-Phe, is located on an exposed β-hairpin loop. It has previously been demonstrated that the macrocyclic octapeptide scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro] is 16-fold less potent than AGRP at the mouse MC4R (mMC4R). Herein it was hypothesized that the Phe position may be substituted to produce more potent and/or selective melanocortin receptor antagonist ligands based on this template. A 10-membered library was synthesized that substituted small (Gly), polar (Ser), acidic (Asp), basic (Lys), aliphatic (Leu, Nle, and Cha), and aromatic (Trp, Tyr, hPhe) amino acids to explore potential modifications at the Phe position. The most potent mMC4R antagonist contained a Nle substitution, was equipotent to the lead ligand 200-fold selective for the mMC4R over the mMC3R, and caused a significant increase in food intake when injected intrathecally into male mice. Three compounds possessed sigmoidal dose-response inverse agonist curves at the mMC5R, while the remaining seven decreased cAMP production from basal levels at a concentration of 100 μM. These findings will add to the knowledge base toward the development of potent and selective probes to study the role of the melanocortin system in diseases of negative energy balance and can be useful in the design of molecular probes to examine the physiological functions of the mMC5R.

The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are established targets to treat diseases of positive and negative energy homeostasis. We previously reported (J. Med. Chem. 60:4342 2017) mixture-based positional scanning approaches to identify dual MC3R agonist and MC4R antagonist tetrapeptides. Herein, 46 tetrapeptides were chosen for MC3R agonist screening selectivity profiles, synthesized, and pharmacologically characterized at the mouse melanocortin-1, −3, −4, and −5 receptors. Substitutions to the tetrapeptide template were selected solely based on MC3R agonist potency from the mixture-based screen. This study resulted in the discovery of compound 42 (Ac-Val-Gln-(pI)DPhe-DTic-NH 2 ), a full MC3R agonist that is 100-fold selective for the MC3R over the µM MC4R partial agonist pharmacology. This compound represents a first-in-class MC3R-selective agonist. This ligand will serve as a useful in vivo molecular probe for the investigation of the roles of the MC3R and MC4R in diseases of dysregulated energy homeostasis.

Antagonist ligands of the melanocortin-3 and -4 receptors (MC3R, MC4R), including agouti-related protein (AGRP), are postulated to be targets for the treatment of diseases of negative energy balance. Previous studies reported the macrocyclic MC3R/MC4R antagonist c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro], which is 250-fold less potent at the mouse (m) mMC3R and 3-fold less potent at the mMC4R than AGRP. Previous studies explored the structure-activity relationships around individual positions in this template. Herein, a multiresidue substitution strategy is utilized, combining the lead sequence with hPhe, Dap, Arg, Ser, and Nle substitutions previously reported. Two compounds from this study (16, 20) contain an hPhe/Ser/Nle substitution pattern, are 3-6-fold more potent than AGRP at the mMC4R and are 600-800-fold selective for the mMC4R over the mMC3R. Another lead compound (21), possessing the hPhe/Arg substitutions, is only 5-fold less potent than AGRP at the mMC3R and is equipotent to AGRP at the mMC4R.

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized β-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding d-isomer(s), generating a 14 compound library. While l-to-d inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.

There is a critical need to find safe therapeutics to treat an increasingly obese population and diseases associated with an imbalance in energy homeostasis. The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) ligands have long been the focus to help scientists understand energy homeostasis and the regulation of feeding behavior. Herein, we use a nanomolar macrocyclic melanocortin receptor agonist ligand MDE6-5-2c (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro) to examine metabolic and energy hemostasis profiles upon intrathecal (IT) administration directly into the spinal cord as compared to intracerebroventricular (ICV) administration directly into the brain. Overall, central ICV administration of MDE6-5-2c resulted in decreased food intake, in a dose-dependent manner, and decreased respiratory exchange ratio (RER). Comparison of IT versus ICV routes of MDE6-5-2c administration resulted in MDE6-5-2c possessing a longer duration of action on both feeding behavior and RER via IT. The C-peptide, ghrelin, GIP, leptin, IL-6, and resistin plasma hormones and biomarkers were compared using IT versus ICV MDE6-5-2c routes of administration. Plasma resistin levels were decreased upon ICV treatment of MDE6-5-2c, as compared to ICV vehicle control treatment. Intrathecal treatment resulted in significantly decreased inflammatory cytokine interleukin-6 (IL-6) levels compared to ICV administration. Investigation of the nonselective MC3R and MC4R macrocyclic agonist MDE6-5-2c molecule revealed differences in food intake, RER, and plasma biomarker profiles

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