B-cell lymphoma 2 (Bcl-2) is a critical antiapoptotic protein and a prime therapeutic target in numerous cancers. The natural product gossypol is a known inhibitor of the Bcl-2 family, but the precise molecular details of its interaction remain elusive, hindering rational drug design efforts. In this study, we employed a comprehensive computational strategy, combining ensemble docking with advanced funnel metadynamics (FM) simulations, to elucidate the binding mechanism of gossypol to Bcl-2 at an atomic level. Our ensemble docking approach successfully predicted a consensus binding pose within the canonical BH3-mimetic groove. Subsequent FM simulations calculated an absolute binding free energy (ΔG) of −6.81 ± 0.86 kcal/mol, which shows reasonable quantitative agreement with the available experimental data. The reconstructed free-energy surface revealed a complex, multistep binding pathway involving a globally stable binding pose and several distinct, metastable intermediate states. Analysis of these states showed that hydrophobic forces are the primary drivers of binding. Furthermore, the interaction is markedly asymmetric; half of the gossypol molecule predominantly anchors the ligand into the P2 and P3 pockets in the most stable binding mode. Crucially, we demonstrate that gossypol binding reduces the overall flexibility of the binding site and that each binding state is characterized by a unique pattern of conformational stabilization across the four pockets. These findings provide an unprecedentedly detailed and dynamic roadmap of the gossypol-Bcl-2 interaction, offering crucial insights for the future structure-based design of next-generation inhibitors.
Zhu et al. (2026) studied this question.