Grafting Hydrophobic Amino Acids Critical for Inhibition of Protein-Protein Interactions on a Cell-Penetrating Peptide Scaffold

Stapled peptides are a promising class of conformationally restricted peptides for modulating protein-protein interactions (PPIs). However, the low membrane permeability of these peptides is an obstacle to their therapeutic applications. It is common that only a few hydrophobic amino acid residues are mandatory for stapled peptides to bind to their target proteins. Hoping to create a novel class of membrane-permeable PPI inhibitors, the phenylalanine, tryptophan, and leucine residues that play a critical role in inhibiting the p53-HDM2 interaction were grafted into the framework of CADY2.a cell-penetrating peptide (CPP) having a helical propensity. Two analogues (CADY-3FWL and CADY-10FWL) induced apoptotic cell death but lacked the intended HDM2 interaction. Pull-down experiments followed by proteomic analysis led to the elucidation of nesprin-2 as a candidate binding target. Nesprin-2 is considered to play a role in the nuclear translocation of beta-catenin upon activation of the Wnt signaling pathway, which leads to the expression of antiapoptosis proteins and cell survival. Cells treated with the two analogues showed decreased nuclear localization of beta-catenin and reduced mRNA expression of related antiapoptotic proteins. These data suggest inhibition of beta-catenin nuclear translocation as a possible mode of action of the described cell-penetrating stapled peptides.