Mapping Protein–Protein Interaction Hotspots and Unveiling a Cryptic Allosteric Pocket in PLK1 PBD via Mixed‐Solvent Molecular Dynamics

Polo-like kinase 1 (PLK1) is a central regulator of mitosis and is frequently overexpressed in diverse human cancers where its elevated levels correlate with poor prognosis. Direct inhibition of the kinase domain often leads to off-target effects due to similarity with other kinases, which has driven efforts to target the polo-box domain (PBD) that mediates protein-protein interactions (PPIs). In this study, we applied mixed-solvent molecular dynamics simulations with amino acid and tripeptide probes to systematically map PPI hotspots on PLK1-PBD. Consistent with prior experimental findings, the canonical PPI site presents challenges for small-molecule drug design. Notably, our analysis revealed a previously uncharacterized allosteric pocket with favorable druggability features. The simulations further indicated that tripeptide probes increase the propensity of pocket opening, highlighting their potential utility for identifying cryptic sites in difficult-to-drug targets. Finally, information-theoretic analysis demonstrated a dynamic coupling between the canonical PPI interface and the newly identified allosteric pocket. Together, these findings provide molecular insights that enable the rational design of next-generation PLK1-PBD inhibitors with potential applications in cancer therapy.