Raman active diyne-girder conformationally constrained p53 stapled peptides bind to MDM2 for visualisation without fluorophores.

Abstract

Peptide stapling is an effective strategy to stabilise α-helical peptides, enhancing their bioactive conformation and improving physiochemical properties. In this study, we apply our novel diyne-girder stapling approach to the MDM2/MDMX α-helical binding region of the p53 transactivation domain. By incorporation of an unnatural amino acid to create an optimal i, i + 7 bridge length, we developed a highly α-helical stapled peptide, 4, confirmed via circular dichroism. This diyne-girder-stapled peptide demonstrated enhanced helicity and nanomolar binding affinity for MDM2, as assessed by fluorescence polarisation. Crucially, peptide 4 exhibited strong selectivity for MDM2, with approximately 100-fold reduced affinity for MDMX. Molecular modeling and docking studies suggested that this selectivity arose from diminished hydrophobic interactions at the MDMX binding site, driven by the diyne-girder's constrained geometry. The use of the diyne-girder, a unique feature amongst stapled peptide analogues, for cellular visualisation using Raman spectroscopy in the "cell-silent" region was explored. This capability potentially offers a novel method for tracking stapled peptides in biological systems without the need for large fluorophores. Overall, peptide 4 represents a promising tool for probing MDM2 activity and a valuable addition to the arsenal of peptide-based therapeutic strategies.