Structure-Activity Relationship Studies of a Macrocyclic AGRP-Mimetic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro] Yield Potent and Selective Melanocortin-4 Receptor Antagonists and Melanocortin-5 Receptor Inverse Agonists That Increase Food Intake in Mice.

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(1)-Arg(2)-Phe(3)-Phe(4)-Asn(5)-Ala(6)-Phe(7)-DPro(8)] is 16-fold less potent than AGRP at the mouse MC4R (mMC4R). Herein it was hypothesized that the Phe(7) 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(7) position. The most potent mMC4R antagonist contained a Nle(7) 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.