153N-6 (H-[Met(5),Pro(6),D-Phe(7),D-Trp(9),Phe(10)]alpha-MSH(5-13)) has emerged 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) [22]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affinities and biologic activities of 153N-6 and 17 selected alpha-MSH analogues at the native MCl receptor expressed by marine B16 melanoma cells. Our intention is to determine the structural requirements for agonism and competitive 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-MSH and the potent synthetic agonist, [Nle(4),D-Phe(7)]alpha-MSH, at the murine MC1-R. However, the K-i of 153N-6 was 439 times higher than that of alpha-MSH and 4475 times higher than that of [Nle(4),D-Phe(7)]alpha-MSH; too high to allow 153N-6 to be considered as a practical antagonist for use in vivo (K-i of 153N-6 = 9.0 X 10(-6) M). Because Met(4) is an important component 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 affinity than 153N-6. The binding affinity of 153N-6 was not significantly different from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-13). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle to the N-terminus of 153N-6 raised the binding affinity by a factor of 46, but 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) derivative of 153N-6 with Ala(4) did not exhibit significantly greater binding affinity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cAMP assay. These data suggest that Met(4) is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MC1-R. Pro(6) and Phe(10) (with respect to alpha-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6. (C) 1999 Elsevier Science Inc. All rights reserved.
