Biological and conformational examination of stereochemical modifications using the template melanotropin peptide, Ac-Nle-c[Asp-His-Phe- Arg-Trp-Ala-Lys]-NH₂, on human melanocortin receptors

Examination of conformationally constrained melanotropin peptides (Ac- Nle⁴-c[Asp⁵-His-Phe⁷ Arg-Trp⁹-Ala-Lys]-NH₂) on four human melanotropin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) resulted in identifying the importance of ligand stereochemistry at positions 5, 7, and 9 for agonist binding affinity and receptor selectivity. A trend in ligand structure- activity relationships emerged for these peptides, with the hMC1R and hMC4R possessing similar tendencies, as did the hMC3R and hMC5R. α-MSH (Ac-Ser- Tyr-Ser-Met⁴-Glu-His-Phe⁷-Arg-Trp-Gly-Lys-Pro-Val-NH₂), NDP-MSH (Ac-Ser- Tyr-Ser-Nle⁴-Glu-His-D-Phe⁷-Arg-Trp-Gly-Lys-Pro-Val-NH₂), and MTII (Ac- Nle⁴-c[Asp⁵,D-Phe⁷,Lys¹⁰]-α-MSH(4-10)-NH₂) were also examined at each of these melanocortin receptors. Interestingly, the linear NDP-MSH possessed greater binding affinity for the hMC3R and hMC5R than did the cyclic analogue MTII. The peptide Ac-Nle-c[Asp-His-Phe-Arg-D-Trp⁹-Ala-Lys]-NH₂ demonstrated the greatest differentiation in binding affinity between the hMC1R and hMC4R (78-fold). Analogue Ac-Nle-c[Asp-His-Phe⁷-Arg-Trp-Ala-Lys]-NH₂ resulted in micromolar binding affinity (or greater) at the hMC3R and hMC5R, demonstrating the importance of D-Phe⁷ for ligand binding potency at these receptors. Ac-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH₂ resulted in loss of binding affinity at the hMC5R, implicating the importance of Nle₄ (or a hydrophobic residue in this position) for binding to this receptor. Ac-Nle-c[D-Asp⁵- His-Phe-Arg-Trp-Ala-Lys]-NH₂ was unable to competitively displace [¹²⁵I]NDP-MSH binding at micromolar concentrations on the hMC3R and hMC5R, suggesting the importance of chirality of Asp⁵ either for ligand-receptor interactions or for orientation of the side chain lactam bridge and the structural integrity of the peptide conformation. Energy calculations performed for these peptides resulted in the identification of a low-energy ligand conformer family that is common to all the ligands. The differences in ligand binding affinities observed in this study are postulated to be a result of different ligand-receptor complexed interactions and not solely to the ligand structure.