Demonstration of a Common DPhe⁷ to DNal(2′)⁷ Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides

Abstract

Melanocortin peptides containing a 3-(2-naphthyl)- d -alanine residue in position 7 (DNal(2′) 7 ), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)- d -alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2′) 7 -containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2′)-Orn-NH2] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2′)-Arg-NH2]

“…Similar antagonist potency at the MC5R (pA 2 = 7.3) was reported from an AGRP-derived macrocyclic octapeptide scaffold (c­[Pro-Arg-Phe-Phe-Dap-Ala-Phe-DPro]) . An alkylthioaryl-bridged cyclic SHU9119 derivative was found to possess antagonist activity (pA 2 = 8.3, antagonist K i = 5 nM) at the hMC5R, similar to the antagonist potency (pA 2 = 8.7, antagonist K i = 2 nM) found at the hMC5R for a cyclic γ-MSH analogue (c­[Nle-Val-DNal(2′)-Arg-Trp-Glu]-NH 2 ), though the receptor constructs assayed in the γ-MSH analogue may have been incorrect . Common to these reported MC5R antagonists is a larger, cyclic structure.…”

“…53 An alkylthioaryl-bridged cyclic SHU9119 derivative was found to possess antagonist activity (pA 2 = 8.3, antagonist K i = 5 nM) at the hMC5R, 54 similar to the antagonist potency (pA 2 = 8.7, antagonist K i = 2 nM) found at the hMC5R for a cyclic γ-MSH analogue (c[Nle-Val-DNal(2′)-Arg-Trp-Glu]-NH 2 ), 55 though the receptor constructs assayed in the γ-MSH analogue may have been incorrect. 56 Common to these reported MC5R antagonists is a larger, cyclic structure. Due to the potential involvement of the MC5R in muscle glucose uptake 57 and acne (as reviewed), 58 the development of short, linear MC5R antagonist ligands may lead to probe compounds that can be used to explore the in vivo functions of the MC5R.…”

Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design

“…The insertion of bulky aromatic amino acid side chains at position seven was recognized early in melanocortin peptide development as conducive to antagonist activity at MC4-R in linear or six- to seven-membered macrocyclic rings. ,,,,− As expected, all PG-934 and SBL-MC-31- derived peptides, featuring DNal(2′) 7 (Table ), showed antagonist activity for Gs-protein-mediated cAMP stimulation at human MC3- and MC4-R (Figures A and S6A,B and Tables and ). In most cases, cAMP accumulation in a cell-based luminescence assay that employs a genetically encoded split firefly luciferase (i.e., GScAMP22F, Promega, Madison, WI) reporter plateaued at 97–100% full inhibition of stimulus by a quasi-maximal (EC 80 to EC 90 ) α-MSH challenge, except SBL-MC-95 exhibiting inhibition of 79.7% of the agonist-elicited response on MC3-R (Tables and ).…”

“…Equally to the receptor: SHU9119 complex, Ca 2+ was coordinated by the receptor acidic E100 2.60 and D122 3.25 side chain carboxyl oxygens and both D126 3.29 side chain oxygens. Additionally, like SHU9119 , PG-934 and SBL-MC-31 coordinated Ca 2+ through the Asp 5 and DNal(2′) 7 backbone carbonyl oxygens (Figures A–C and A,B and Table S4), highlighting the stabilizing effect of many macrocyclic rings on Ca 2+ binding, regardless of their agonist or antagonist activity on MC4-R. For instance, SHU9119 has partial agonist activity on MC1-R, but the same coordination network for this ligand was still described in the MC1-R: SHU9119 (PDB: 7F4I) and MC1-R:Nle 4 -DPhe 7 α-MSH (PDB: 7F4F) complexes and even linear or cyclic peptides with MC2-R, (PDB: 8GY7) MC3-R (PDB: 8IOC), and MC5-R (PDBs: 8INR and 8IOD). , …”

Melanocortin-5 Receptor: Pharmacology and Its Regulation of Energy Metabolism

“…However, these synthetic ligands can also effectively activate (or antagonize, as in the case of SHU9119) the other MCR subtypes, suggesting that they do not exhibit good selectivity for MC5R. Subsequently, agonists highly specific to MC5R were developed, including PG-901, PG-911, OBP-MTII (Oic 6 , D-4,4′-Bip 7 , Pip 8 -MTII), and others [ 99 , 100 , 101 ].…”