Structure-Guided Grafting of Cell-Penetrating Motifs into Stapled Peptides for Intracellular PPI Inhibition

Abstract Protein–protein interactions (PPIs) are promising drug targets, yet peptide modulation of intracellular PPIs is limited by poor membrane permeability. Although conjugation with cell-penetrating peptides (CPPs) improves delivery, it often increases molecular weight, induces nonspecific interactions, and compromises target binding. Here, we present a CPP grafting strategy that integrates membrane permeability into peptide design while preserving binding capability. Based on computational identification of hotspot residues, CPP motifs were embedded into regions dispensable for binding. Applied to the MDM2/p53 interaction, the CPP-grafted stapled peptide St3-R showed enhanced cellular uptake and improved intracellular inhibitory activity compared with CPP-conjugated analogues. X-ray crystallography confirmed the preservation of key hydrophobic hotspot residues after grafting. The strategy was also validated in the β-catenin/TCF system, where CPP-grafted peptides retained binding affinity, suppressed Wnt signaling, and inhibited cancer cell proliferation. This approach provides a structure-guided platform for intracellular PPI-targeting peptides.