Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine

U. G. Sahm, G. W. Olivier, S. K. Branch, S. H. Moss, C. W. Pouton

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were 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 results using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual residues. 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 response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe7, Arg8, and Trp9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.
Original languageEnglish
Pages (from-to)1297-1302
Number of pages6
JournalPeptides
Volume15
Publication statusPublished - 1994

Keywords

  • Alanine/chemistry Amino Acid Sequence Animals Binding, Competitive Melanoma, Experimental/metabolism Mice Molecular Sequence Data Monophenol Monooxygenase/metabolism Radioligand Assay Receptors, Corticotropin/metabolism Receptors, Melanocortin Structure-Activity Relationship Tumor Cells, Cultured/metabolism alpha-MSH/*analogs & derivatives/chemical synthesis/metabolism

Cite this

Sahm, U. G., Olivier, G. W., Branch, S. K., Moss, S. H., & Pouton, C. W. (1994). Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine. Peptides, 15, 1297-1302.
Sahm, U. G. ; Olivier, G. W. ; Branch, S. K. ; Moss, S. H. ; Pouton, C. W. / Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine. In: Peptides. 1994 ; Vol. 15. pp. 1297-1302.
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abstract = "The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were 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 results using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual residues. 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 response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe7, Arg8, and Trp9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.",
keywords = "Alanine/chemistry Amino Acid Sequence Animals Binding, Competitive Melanoma, Experimental/metabolism Mice Molecular Sequence Data Monophenol Monooxygenase/metabolism Radioligand Assay Receptors, Corticotropin/metabolism Receptors, Melanocortin Structure-Activity Relationship Tumor Cells, Cultured/metabolism alpha-MSH/*analogs & derivatives/chemical synthesis/metabolism",
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Sahm, UG, Olivier, GW, Branch, SK, Moss, SH & Pouton, CW 1994, 'Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine' Peptides, vol. 15, pp. 1297-1302.

Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine. / Sahm, U. G.; Olivier, G. W.; Branch, S. K.; Moss, S. H.; Pouton, C. W.

In: Peptides, Vol. 15, 1994, p. 1297-1302.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine

AU - Sahm, U. G.

AU - Olivier, G. W.

AU - Branch, S. K.

AU - Moss, S. H.

AU - Pouton, C. W.

N1 - M1 - 7 Sahm, U G Olivier, G W Branch, S K Moss, S H Pouton, C W eng Research Support, Non-U.S. Gov't 1994/01/01 Peptides. 1994;15(7):1297-302.

PY - 1994

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N2 - The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were 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 results using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual residues. 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 response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe7, Arg8, and Trp9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

AB - The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were 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 results using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual residues. 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 response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe7, Arg8, and Trp9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

KW - Alanine/chemistry Amino Acid Sequence Animals Binding, Competitive Melanoma, Experimental/metabolism Mice Molecular Sequence Data Monophenol Monooxygenase/metabolism Radioligand Assay Receptors, Corticotropin/metabolism Receptors, Melanocortin Structure-A

M3 - Article

VL - 15

SP - 1297

EP - 1302

JO - Peptides

JF - Peptides

SN - 0196-9781

ER -