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

153N-6 (H-[Met⁵,Pro⁶,D-Phe⁷,D-Trp⁹,Phe¹⁰]α-MSH((5-13))) has emerged as the most potent antagonist of α-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on α-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 α-MSH analogues at the native MC1 receptor expressed by murine 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 α-MSH and the potent synthetic agonist, [Nle⁴,D-Phe⁷]α-MSH, at the murine MC1-R. However, the K(i) of 153N-6 was 439 times higher than that of α-MSH and 4475 times higher than that of [Nle⁴,D-Phe⁷]α-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 Met⁴ is an important component of α-MSH binding at the MC1-R, we investigated α-MSH((1-13)) and α-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 α-MSH((5-13)), but it was 232 times lower than α-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 α-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length α-MSH((1-13)) derivative of 153N-6 with Ala⁴ 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 Met⁴ is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MC1-R. Pro⁶ and Phe¹⁰ (with respect to α-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6. Copyright (C) 1999 Elsevier Science Inc.