Evaluation of Small Molecule Binding to the Polo-Box Domain of PLK1 at the Molecular Level

The design and development of Polo-like kinase 1 (PLK1) inhibitors have gained prominence as potential, less toxic alternative for cancer treatment. Specifically, the polo-box domain (PBD) of PLK1 is a promising target since its inhibition can disrupt the specific localization and substrate interactions of PLK1 without directly inhibiting its kinase activity. Therefore, selectively interfering with PLK1 function, such as in cancer proliferation, is plausible while minimizing the impact on normal cellular functions. This study provides a molecular-level evaluation of small-molecule binding to the PBD, identifying key residues and interactions that govern ligand recognition across seven distinct binding sites, including both experimentally established pockets (BS1-BS3) and newly identified cavities (BS4–BS7). The binding mechanisms of eight nonpeptidic inhibitors at these binding sites were studied through molecular dynamics (MD) simulations totaling 11.6 [Formula: see text]s and adaptive steered MD (ASMD) simulations totaling 1.6 [Formula: see text]s. The ligands, thymoquinone (TQ), four Poloxin derivatives, enantiomers (–)–9* and ([Formula: see text]–9*, and Allopole-A, were selected based on their reported inhibitory activity, structural diversity, and potential for specific interactions with PBD. Our findings reveal that ligand binding is stabilized by extensive hydrogen bonding (HB) and hydrophobic interactions with residues such as Trp414 in the phosphopeptide pocket. Also, we observed that stereochemistry and minor structural modifications significantly impact ligand–protein interactions within the PLK1-PBD. Enantiomers like (–)–9* and ([Formula: see text]–9* exhibit distinct binding behaviors, while structural variations among Poloxin derivatives highlight the importance of functional group placement and electronic effects. ASMD simulations further revealed site-specific flexibility and ligand-induced conformational changes. Notably, Allopole-A exhibits robust allosteric inhibition by stabilizing the W-F pocket and inducing structural distortion that disrupts phosphopeptide binding (PMF difference [Formula: see text] 2.7 kcal/mol). In contrast, Poloxin and (–)–9* function as competitive inhibitors, slowing phosphopeptide dissociation without significantly altering the PBD’s conformational dynamics (PMF differences of 4.9 kcal/mol and 7.3 kcal/mol, respectively). These insights into ligand-PBD interactions and binding mechanisms may inform future efforts to develop PLK1 inhibitors with improved specificity, including nonpeptidic compounds acting through alternative mechanisms such as allosteric or competitive inhibition.