Synthesis of 153N-6 analogues and structure-function analysis at murine melanocortin-1 (MC1) receptors

153N-6 (H-[Met(5),Pro(6),D-Phe(7),D-Trp(9),Phe(10)]alpha-MSH(5-13)) has eme rged as the most potent antagonist of alpha-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on alpha-MSH(5-13) [2 2]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affi nities and biologic activities of 153N-6 and 17 selected alpha-MSH analogue s at the native MCl receptor expressed by marine B16 melanoma cells. Our in tention is to determine the structural requirements for agonism and competi tive antagonism of melanocortin activity at the MC1-R and to discover more potent antagonists. 153N-6 was able to inhibit the action of native alpha-M SH and the potent synthetic agonist, [Nle(4),D-Phe(7)]alpha-MSH, at the mur ine MC1-R. However, the K-i of 153N-6 was 439 times higher than that of alp ha-MSH and 4475 times higher than that of [Nle(4),D-Phe(7)]alpha-MSH; too h igh to allow 153N-6 to be considered as a practical antagonist for use in v ivo (K-i of 153N-6 = 9.0 X 10(-6) M). Because Met(4) is an important compon ent of alpha-MSH binding at the MC1-R, we investigated alpha-MSH(1-13) and alpha-MSH(4-13) analogues to produce compounds with higher MC1-R-binding af finity than 153N-6. The binding affinity of 153N-6 was not significantly di fferent from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-1 3). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle t o the N-terminus of 153N-6 raised the binding affinity by a factor of 46, b ut this and all full-length alpha-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length alpha-MSH(1-13) derivativ e of 153N-6 with Ala(4) did not exhibit significantly greater binding affin ity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cA MP assay. These data suggest that Met(4) is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at t he MC1-R. Pro(6) and Phe(10) (with respect to alpha-MSH) were found to be t he most influential substitutions that determined the antagonist activity o f 153N-6. (C) 1999 Elsevier Science Inc. All rights reserved.