No abstract

To elucidate the molecular basis of the interaction of the native dodecapeptide gamma-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced l-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of gamma-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in gamma-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of gamma-MSH.

The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.

It has been shown by extensive studies that alpha-MSH bioactivity is critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, however with poor selectivity for the human MC3R-MC5R. The structure-activity relationships study here is aimed at identifying lead structures or templates of this core sequence by the use of different conformational constraints that might impart changes in its topography and thus promote differences in potency and selectivity at these receptors. Our peptide library consists of a novel series of cyclic alpha-MSH analogues that have disulfide bridges between Cys or Cys-like residues at positions 4 and 10, giving rise to 23-membered rings fused at the C-terminal end with the C-terminal fragment of beta-MSH (Pro-Pro-Lys-Asp). While such constraints of the peptide backbone with disulfide bridges of different chirality affect potency and selectivity at these receptors, further changes in the hydrophobicity at position 7 with either a D-Phe or D-Nal(2') and replacement of a His with a Pro in position 6 cause additional effects. Thus, the most interesting lead compounds that emerged from this study are (1) compound 5, Ac-c[Cys-Glu-His-D-Phe-Arg-Trp-D-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 10 nM), which is the first potent and highly selective antagonist ligand for the hMC5R (560-fold vs the MC3R and 1000-fold vs the MC4R); (2) compound 7, Ac-c[Cys-Glu-Pro-D-Nal(2')-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 31 nM), which is a highly selective antagonist analogue for the MC3R (560-fold vs the hMC4R and about 3000-fold vs the hMC5R; and (3) compound 9, Ac-c[Pen-Glu-His-D-Nal(2')-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 3 nM), which is more potent than 7 at the MC3R but not as selective.

It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in alpha-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in gamma-MSH, two different series of gamma-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native gamma-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr-Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH(2) with Gly(4)) is more important for selectivity. The 2nd series is made of d-2-naphthylalanine (d-Nal(2')) scan of the gamma-MSH sequence at position 6 and 8 and the replacement of His(5) with Pro (analogue 13). Analogue 12, H-Tyr-Val-Nle-Gly-His-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr-Val-Nle-Gly-Pro-d-Nal(2')-Arg-Trp-Asp-Arg-Phe-Gly-NH(2), which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores.