Synthesis and characterization of a Eu-DTPA-PEGO- MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low mu M affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-DPhe-Arg-Trp-NH2, exhibited a K-d for hMC4R of 9.1 +/- 1.4 mu M, approximately 10-fold lower affinity than the parental ligand. The labeled MSH( 4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH( 4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-alpha-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects. (C) 2010 Elsevier Ltd. All rights reserved.