STRUCTURE-BASED SEARCH FOR PEPTIDE LIGANDS THAT CROSS-REACT WITH MELANOCORTIN RECEPTORS

Purpose. To define sequence motifs that can be used to identify peptid e ligands of the melanocortin receptor (MCR). Methods. Screening of co mbinatorial libraries has led to identification of D-Trp-Nle-NH2 (Nle, norleucine) and D-TrpArg-NH2 as the smallest structures known to anta gonize the amphibian MCR (1). As the basis of a search paradigm, pepti de-ligands containing these or similar motifs within their larger prim ary structure were examined for ability to antagonize amphibian and re combinant human MCRs. Compounds examined include analogs of substance P, leutinizing-hormone releasing-hormone, endothelin, neurotensin, and opioid-somatostatin. Results. Of seven compounds tested containing th e predetermined search motif D-Trp-AA(x) (where AA(x) is Arg, Leu, Ile , or ne), six were found to have previously unrecognized antagonist ac tivity at the amphibian MCR (K-d 30 to 5000 nM). In contrast, of 14 si milar control peptides lacking the D-Trp-AA(x), search motif, only som atostatin displayed measurable antagonist potency. The anticancer pept ide, [Arg(8), D-Trp(7,9), N-methyl-Phe(8)]-substance P, was the most p otent of the motif-containing peptides with a K-d of 31 nM. The mu-opi oid antagonist D-Phecyclic[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH2 (CTAP) a lso blocked the amphibian MCR (K-d 1 mu M), but the related mu-antagon ist CTOP, different only by only by substitution of Arg with ornithine within the search motif, was found to agonize the amphibian MCR (EC(5 0) 67 nM). CTAP and the anticancer peptide were also tested on human M CRs (hMCRs); while CTAP competed with alpha-MSH at the hMCl receptor, the anticancer peptide had no effect or was slightly stimulatory. Conc lusions. We have identified dipeptide motifs that help distinguish ant agonist ligands of the amphibian MCR from ligands known to interact wi th other, G-protein coupled receptors. ?his approach might be generall y applicable if motifs can identified for other receptors and their su btypes. In studies employing CTAP and CTOP, analogs previously conside red highly selective for the mu-opioid receptor, crossreaction with MC Rs must be considered.