Elucidating the Covalent Binding of Ottelione A and Withaferin A at the Colchicine Site of β‑Tubulin.

Ottelione A (OTT) and withaferin A (WIT) are natural products known to induce microtubule depolymerization by covalently modifying β-tubulin within the colchicine binding site (COLbs). Experimental evidence suggests that both compounds primarily react with βCys239 through Michael-type additions. In this work, orbital-weighted dual descriptor analysis, relative stability of methyl thiolate adducts, ensemble covalent docking, and molecular dynamics simulations were employed to investigate their reactivity and covalent binding modes. While initial reactivity analysis identified multiple electrophilic centers in both ligands, OTT shows a thermodynamic preference for 1,6-addition products due to hyperconjugative stabilization, whereas WIT favors 1,4-addition products over endergonic 1,2-addition. Docking and molecular dynamics simulations are consistent with these observations, as OTT preferentially forms 1,6-Michael adducts within the COLbs, while WIT forms 1,4-Michael adducts and can adopt an alternative conformation oriented toward the αβ-tubulin interface. Notably, this pose promotes displacement of the β-T7 loop and increased solvent exposure of the hydrophobic COLbs pocket, suggesting a potential structural basis for tubulin destabilization. Overall, this study provides molecular insight into the covalent interaction of OTT and WIT with β-tubulin and suggests structural features underlying their microtubule-destabilizing activity.