Multiresidue Tetrapeptide Substitutions Yield a 140-fold Selective Melanocortin-3 over Melanocortin-4 Receptor Agonist

Abstract

The five melanocortin receptors regulate numerous physiological functions. Although many ligands have been developed for the melanocortin-4 receptor (MC4R), the melanocortin-3 receptor (MC3R) has been less-well characterized, in part due to the lack of potent, selective tool compounds. Previously an Ac-His-Arg-(pI)­DPhe-Tic-NH2 scaffold, inverting the Phe-Arg motif of the native melanocortin signal sequence, was identified to possess mMC3R over mMC4R selective agonist activity. In this study, a library of 12 compounds derived from this scaffold was synthesized and assayed at the mouse melanocortin receptors (MCRs), utilizing substitutions previously shown to increase mMC3R agonist potency and/or selectivity. One compound (8, Ac-Val-Gln-DBip-DTic-NH2) was identified as greater than 140-fold selective for the mMC3R over the mMC4R, possessed 70 nM potency at the mMC3R, and partially stimulated the mMC4R at 100 μM concentrations without antagonist activity. This pharmacological profile may be useful in developing new tool and therapeutic ligands that selective signal through the MC3R.

Incorporation of Indoylated Phenylalanine Yields a Sub-Micromolar Selective Melanocortin-4 Receptor Antagonist Tetrapeptide

“…The stereochemistry at this position within the scaffold has not previously been explored, with prior reports always utilizing a d -amino acid. 27 , 41 − 43 The original mixture-based positional scan identified (pCl)DPhe and (pI)DPhe as the substitutions at the third position with the highest percentage activity in the initial screening (20 and 26%, respectively) for mMC3R activity, with minimal MC3R activity reported for the (pCl)Phe and (pI)Phe l -isomers (2 and −1%, respectively). The data herein supports the mixture-based positional scan results of the importance of d -stereochemistry at the third position for melanocortin agonism within this scaffold.…”

“…Both 4 and 5 also possessed micromolar mMC4R antagonist potency (pA 2 = 5.5 and 5.2, respectively). Previous studies examining similar tetrapeptides reported similar pharmacological activities at the mMC1R, mMC3R, mMC4R, and mMC5R when the imino acids Tic or DTic were substituted at the fourth position. , Another study showed that substitution of the α-amino acids Nal(2′) or DNal(2′) at the fourth position of the Ac-Arg-Arg-(pI)­DPhe-Xxx scaffold resulted in similar agonist potencies at the mMC1R and mMC5R and similar antagonist potencies at the mMC4R, and both could interact with MC3R, although a DNal(2′) substitution resulted in agonist activity and Nal(2′) substitution possessed antagonist activity . The results support the present study that indicates that the fourth position within the scaffold can tolerate both l and d isomers, although the decreased potencies without corresponding gains in selectivity suggest that additional stereochemical bulk was not beneficial in ligand design.…”

“…Compounds substituted with indoylated d -phenylalanine at the third position ( 8 and 9 ) possessed partial agonist activity at the mMC1R, mMC3R, and mMC5R (with compound 8 possessing full agonist efficacy at the mMC5R), and both were micromolar potent antagonists at the mMC4R (pA 2 = 5.7 and 5.5 for 8 and 9 , respectively). The stereochemistry at this position within the scaffold has not previously been explored, with prior reports always utilizing a d -amino acid. ,− The original mixture-based positional scan identified (pCl)­DPhe and (pI)­DPhe as the substitutions at the third position with the highest percentage activity in the initial screening (20 and 26%, respectively) for mMC3R activity, with minimal MC3R activity reported for the (pCl)­Phe and (pI)­Phe l -isomers (2 and −1%, respectively). The data herein supports the mixture-based positional scan results of the importance of d -stereochemistry at the third position for melanocortin agonism within this scaffold.…”