Revealing the Binding Mechanism of Gossypol on Bcl‑2 via Funnel Metadynamics Simulations

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.