SYNTHESIS AND BIOLOGICAL EVALUATION OF ALPHA-MSH ANALOGS SUBSTITUTED WITH ALANINE

The influence of single amino acid replacements by alanine on the bind ing affinity and biological activity of alpha-MSH in B16 murine melano ma cells has been studied systematically. alpha-MSH analogues were syn thesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells we re determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier r esults using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual resi dues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4-9 forming a core sequence required for receptor binding and triggering of the biological respons e. It was observed that replacement of the glutamic acid residue in po sition 5 was possible without loss of affinity or activity, whereas re placement of Met(4) resulted in a 100-fold loss of binding affinity an d biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe(7), Arg(8) , and Trp(9) being the most sensitive to replacement by alanine. Gener ally, there was a rank correlation between binding affinity and tyrosi nase stimulation within the group of analogues studied. Tyrosinase act ivity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.