Amlodipine besylate, a Dihydropyridine derivative as a Tubulin polymerization inhibitor.

Cancer remains a critical global health burden owing to multidrug resistance. Most anticancer drug candidates fail in clinical trials because of safety and efficacy concerns. Drug repurposing, which uses clinically approved medications for new applications, mitigates this risk. In our previous study, we reported that amlodipine besylate (AB), a dihydropyridine derivative and a third-generation calcium channel blocker (CCB), exhibited cytotoxic activity toward cervical cancer (HeLa) cells (IC₅₀ ≈ 10 µM) by disrupting microtubule architecture. Thus, in this study, we aimed to validate the interaction between AB and purified tubulin. Binding affinity studies showed that AB binds to tubulin with an apparent dissociation constant (KD) of ~ 1.2 µM. DTNB kinetic assay, ANS binding assay, and CD spectroscopy data indicated that AB binding induces a conformational change in tubulin upon ligand interaction. In vitro tubulin polymerization assay revealed that AB inhibits tubulin polymerization with an IC₅₀ value of 8.97 ± 0.55 µM. Competition experiments with podophyllotoxin (a colchicine site binding agent) did not affect AB binding to tubulin, whereas vinblastine reduced AB–associated fluorescence, suggesting reduced binding of AB. Molecular docking suggested that AB interacts within the vinblastine-binding domain with less favorable predicted binding energy compared to vinblastine. Molecular dynamics simulation further predicted that the initial docking pose of AB within the vinblastine-binding domain was transient and showed positional relocation, suggesting that vinblastine affects AB binding possibly via conformational modulation rather than direct competition. Thus, the molecular structure of AB may serve as a valuable scaffold for the synthesis of new derivatives with enhanced anticancer activity.