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

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 β-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 β-catenin and reduced mRNA expression of related antiapoptotic proteins. These data suggest inhibition of β-catenin nuclear translocation as a possible mode of action of the described cell-penetrating stapled peptides.