Structure-based search for peptide ligands that cross-react with melanocortin receptors.

PURPOSE

To define sequence motifs that can be used to identify peptide ligands of the melanocortin receptor (MCR).

METHODS

Screening of combinatorial libraries has led to identification of D-Trp-Nle-NH2 (Nle, norleucine) and D-Trp-Arg-NH2 as the smallest structures known to antagonize the amphibian MCR (1). As the basis of a search paradigm, peptide-ligands containing these or similar motifs within their larger primary structure were examined for ability to antagonize amphibian and recombinant human MCRs. Compounds examined include analogs of substance P, leutinizing-hormone releasing-hormone, endothelin, neurotensin, and opioid-somatostatin.

RESULTS

Of seven compounds tested containing the predetermined search motif D-Trp-AAx (where AAx is Arg, Leu, Nle, or Ile), six were found to have previously unrecognized antagonist activity at the amphibian MCR (Kd 30 to 5000 nM). In contrast, of 14 similar control peptides lacking the D-Trp-AAx search motif, only somatostatin displayed measurable antagonist potency. The anticancer peptide, [Arg8, D-Trp7.9, N-methyl-Phe8]-substance P, was the most potent of the motif-containing peptides with a Kd of 31 nM. The mu-opioid antagonist D-Phe-cyclic[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH2 (CTAP) also blocked the amphibian MCR (Kd 1 microM), but the related mu-antagonist CTOP, different only by only by substitution of Arg with ornithine within the search motif, was found to agonize the amphibian MCR (EC50 67 nM). CTAP and the anticancer peptide were also tested on human MCRs (hMCRs); while CTAP competed with alpha-MSH at the hMC1 receptor, the anticancer peptide had no effect or was slightly stimulatory.

CONCLUSIONS

We have identified dipeptide motifs that help distinguish antagonist ligands of the amphibian MCR from ligands known to interact with other G-protein coupled receptors. This approach might be generally applicable if motifs can identified for other receptors and their subtypes. In studies employing CTAP and CTOP, analogs previously considered highly selective for the mu-opioid receptor, cross-reaction with MCRs must be considered.