Elucidation of a Novel Dual Binding Site on Tubulin: Theoretical Insights and Prospective Hybrid Inhibitors

<b>Background/Objectives:</b> Microtubule-targeting agents remain foundational components of anticancer chemotherapy, yet their clinical utility is constrained by resistance and toxicity. <b>Methods:</b> Here, we present a theoretical exploration of a plausible "dual" binding pocket that spans the α-tubulin pironetin site and the inter-subunit todalam site. Eight virtual chimeric ligands, each merging key pharmacophoric elements of pironetin and todalam, were constructed and covalently docked to Cys316 of α-tubulin. <b>Results:</b> Covalent docking followed by 200 ns all-atom molecular dynamics simulations revealed that two derivatives (compounds <b>4</b> and <b>8</b>) stably occupy the merged cavity, simultaneously anchoring in the pironetin region via Michael addition and in the todalam region via π-stacking and hydrogen bonding. These hybrids preserved the critical hydrogen-bonding networks of both parent ligands and exhibited low ligand RMSD values (~1.5 Å) and compact radii of gyration throughout the simulations, indicating a tight, persistent binding. Estimated HYDE affinities of 1.5 µM for compound 4 and 17.6 µM for compound <b>8</b>, calculated with SeeSAR, suggest that covalent engagement can compensate for moderate non-covalent binding scores. <b>Conclusions:</b> In summary, our results provide compelling grounds for developing a new class of α-tubulin inhibitors that engage the hybrid pocket, laying a foundation for the structure-guided synthesis of first-in-class dual-site compounds capable of overcoming resistance to conventional microtubule-targeting drugs.