Therapeutic targeting of cancer-associated PKM2 mutants by shikonin via mutation-induced pocket recognition.

Pyruvate kinase (PKM2) is a key glycolytic enzyme involved in cancer metabolic reprogramming and frequently harbors oncogenic missense mutations that alter its structural and functional dynamics. Here, we investigate the molecular basis of recognition of the anti-cancer drug shikonin in wild type (WT) and six PKM2 mutants. Molecular docking identified three mutants (R246S, P117L, and H464A) exhibiting favourable binding of shikonin within a newly identified pocket adjacent to the allosteric site, while the WT accommodated shikonin in the known active site pocket, consistent with previous literature reports. Subsequently, 1 µs MD simulations confirmed reduced conformational fluctuations, higher values and persistent residue contacts in these mutants, whereas the other three mutants (K367M, R399E, and R455Q) displayed disrupted pocket integrity, lower values and unstable ligand binding. Rg, PCA, Fpocket program and residue communication network analyses revealed that the favourable binding mutants maintain a compact pocket architecture stabilized through efficient short-range allosteric coupling between the mutation sites and pocket residues and druggability scores greater than 0.5, while weak-binding mutants show weakened long-range communication pathways. Interaction profiling further identified stable hydrogen bonding and stacking interactions supported by favourable interaction energies. Overall, this study demonstrates that PKM2 mutations dynamically remodel the binding pocket and allosteric communication to facilitate shikonin recognition, highlighting the newly identified pocket as a promising therapeutic site for future PKM2 targeted drug design.