Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists

Abstract

Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His⁶-d-Nal(2')⁷-NMe-Arg⁸-Trp⁹-Lys]-NH₂ (15) and Ac-Nle-c[Asp-His⁶-d-Nal(2')⁷-NMe-Arg⁸-NMe-Trp⁹-NMe-Lys]-NH₂ (17). It is known that the pharmacophore (His⁶-DNal⁷-Arg⁸-Trp⁹) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal⁷-Arg⁸. The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp⁹ side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg⁸ and Trp⁹ side chains are involved in a majority of the interactions with the receptor. While Arg⁸ forms polar contacts with D154 and D158 of hMC3R, Trp⁹ utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp⁹-hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

Figure 1

Model for receptor–ligand complexation in hMC3R activation. Two-dimensional representation of a proposed three-dimensional model illustrating the synthetic melanocortin NDP-α-MSH bound to hMC3R. Two major receptor binding sites are hypothesized, with the first being a predominantly ionic pocket formed by D154 and D158, and the second a hydrophobic pocket formed by aromatic residues in TM6.^–

Figure 2

NMR structures of hMC3R peptides 15 and 17. Stereo views of the NMR structure of peptides 15 (a, pink); 17 (b, green); and the overlay of the β-turn like His⁶-nal⁷-Arg⁸-Trp⁹ region from peptides 15 and 17 (c). Ribbons along backbones highlight secondary structural features. For clarity, nonpolar hydrogens in a and b are shown, whereas all the hydrogens and the main chain in c are not shown.

Figure 3

Backbone conformations of peptides 15 and 17 are largely consistent with hMC3R ligands MTII and SHU9119, with a few notable differences. Ramachandran plots that represent the (Φ,Ψ) dihedral space of constituent amino acids and corresponding secondary structural information, generated for (a) peptides 15 and 17 based on their NMR structures and (b) for peptides MTII and SHU9119. (c) Graph showing the site-specific variations in the dihedral space.

Figure 4

Docked ligands interact with hMC3R residues that are crucial for ligand binding and activity. (a) Contact probability (calculated as percentage of docking clusters with ligand atoms within 4.0 Å of specified hMC3R residue) of MTII to interact with functionally important residues in hMC3R. (b) Contact probability of SHU9119 with residues in hMC3R. (c) Contact probability for peptide 15. (d) Contact probability for peptide 17.

Figure 5

N-methylation modulates localized effectiveness of binding in SHU9119 analogues. (a) Per-residue number of contacts between MTII and hMC3R in docking studies. Average number of contacts is defined as total number of interactions within 4.0 Å between ligand atoms and any atom in hMC3R averaged over 18 docking clusters. Residue seven is DPhe in MTII (a) and DNal in peptide 15 (c). (b) Per-residue number of contacts between SHU9119 and hMC3R. (c) Per-residue number of contacts between peptide 15 and hMC3R. (d) Per-residue number of contacts between peptide 17 and hMC3R.

Figure 6

Polar and π–π interactions stabilize binding of ligands to the hMC3R binding pocket. (a) Snapshot of lowest-energy binding complex between MTII and hMC3R. (b) Snapshot of lowest-energy binding complex between SHU9119 and hMC3R. (c) Snapshot of lowest-energy binding complex between peptide 17 and hMC3R. (d) Snapshot of the lowest-energy binding complex between peptide 15 and hMC3R. Sticks, docked ligands; ball and sticks, hMC3R amino acid residues; dashes, nonbonded interactions between ligand and receptor <5 Å; gray ribbons, cartoon representation of hMC3R; red, oxygen atoms; blue, nitrogen; white, hydrogen; green, carbons in hMC3R; orange, MTII carbons; cyan, SHU9119 carbons; purple, peptide 17 carbons; brown, peptide 15 carbons. All complexes were rendered in PyMOL.