Rational design of thiazolidine-2,4-dione-integrated combretastatin A-4 analogues as potent and selective tubulin-targeting agents for triple-negative breast cancer.
Abstract
Microtubule-targeting agents remain among the most effective chemotherapeutics for the treatment of cancer. Inspired by the natural tubulin polymerization inhibitor combretastatin A-4 (CA-4), a series of novel thiazolidine-2,4-dione derivatives was designed, synthesized, and evaluated as potential tubulin-targeting anticancer agents. The cytotoxic activities of the compounds were assessed against the MDA-MB-231, while human umbilical vein endothelial cells (HUVEC) were employed to evaluate selectivity. Compounds 2 and 3 emerged as the most active members of the series (IC50 = 3.60 and 3.71 μM, respectively), accompanied by remarkably high selectivity indices of 49.7 and 51.3. To elucidate their mechanism of action, compounds 2 and 3 were further evaluated in an in vitro tubulin polymerization assay. Compound 3 displayed the strongest inhibitory activity (IC50 = 1.07 μM), surpassing the reference inhibitor CA-4 (IC50 = 2.73 μM). Flow cytometric analysis showed that compound 3 induced G2/M cell-cycle arrest, increasing the G2/M population from 21.8% to 30.8%, and markedly promoted apoptosis, with total apoptotic cells increasing from 0.13% to 43.9% in MDA-MB-231 cells. Molecular docking and MM-GBSA calculations revealed favorable binding within the colchicine-binding site of tubulin, with compounds 2 and 3 exhibiting substantially stronger predicted binding affinities (ΔG = -89.53 and -92.86 kcal/mol, respectively) than CA-4 (ΔG = -70.08 kcal/mol). Molecular dynamics simulations confirmed the stability of the ligand-protein complexes throughout the simulation period, supporting the proposed binding mode. Furthermore, in silico ADME analysis suggested favorable drug-likeness, high predicted oral absorption, and improved pharmacokinetic characteristics compared with paclitaxel. The present study identifies compounds 2 and 3 as promising lead candidate that combines potent and selective antiproliferative activity, strong tubulin polymerization inhibition, and favorable binding characteristics.