Identification of Novel Potential Tubulin-Targeting Heterocyclic Compounds via Integrated Virtual Screening and In Vitro Studies

Background: The development of novel tubulin-targeting agents remains a promising strategy in anticancer drug design. In the present study, novel heterocyclic tubulin-targeting compounds were identified using a complex in silico/in vitro workflow. Methods: An in-house library of approximately 8000 structurally diverse heterocyclic compounds was screened for antiproliferative activity, yielding 260 experimentally validated hits. These compounds were docked against the colchicine, vinca, and taxane binding sites of tubulin. Selected compounds were also tested for their effects on cellular β-tubulin protein levels in Jurkat cells. Results: Docking analysis indicated that 42 compounds matched or exceeded colchicine’s binding energy, 3 compounds were comparable to vinblastine, and 18 compounds surpassed paclitaxel, suggesting the colchicine and taxane sites as preferred targets. Five top-scoring compounds (Les-6370, Les-4272, Les-4309, Les-4403, and Les-1419) were further analyzed via molecular dynamics simulations, confirming stable binding—particularly at the colchicine site—and favorable hydrogen-bonding and lipophilic interactions. At 10 µM, Les-1419 and Les-4309 reduced total cellular β-tubulin protein levels in Jurkat cells by 57.73% and 54.31%, respectively, relative to the effect observed for the reference compound Combretastatin A-4. Conclusions: This study resulted in the identification of novel starting scaffolds with favorable tubulin interaction activity and predicted drug-like properties, representing structurally novel parent structures and serving as promising starting points for further structural optimization and analogue development.