Gastrointestinal stromal tumors (GISTs) are primarily treated with tyrosine kinase inhibitors (TKIs) such as imatinib. However, the development of drug resistance remains a critical clinical challenge, highlighting the urgent need for novel therapeutic agents with alternative mechanisms of action. This study aimed to identify and characterize new small molecules effective against imatinib-sensitive and resistant GISTs.

A library of 27 benzo[d]imidazo[2,1-b]thiazole derivatives was synthesized and screened for cytotoxicity against four GIST cell lines, including imatinib-sensitive (GIST-T1, GIST-882) and imatinib-resistant (GIST-48, GIST-48B) models. The most effective compound, MBT3T, was further evaluated for safety on healthy cells (PBMCs, fibroblasts) and mechanism of action using computational docking, immunofluorescence, and flow cytometry. Efficacy was validated both in 3D bioprinted tumor models and in vivo using zebrafish xenografts.

MBT3T exhibited potent nanomolar cytotoxicity across all GIST cell lines while maintaining a favorable safety profile on healthy cells. Mechanistic studies revealed that MBT3T acts independently of the KIT signaling pathway, targeting tubulin at the colchicine-binding site. This interaction caused microtubule disassembly, leading to G2/M cell cycle arrest and caspase-mediated apoptosis. In in vivo zebrafish assays, MBT3T significantly reduced tumor growth in both imatinib-sensitive and resistant xenografts without inducing systemic toxicity.

We identified MBT3T as a novel, selective tubulin polymerization inhibitor. Its ability to bypass TKIs resistance mechanisms suggests it may represent a promising therapeutic candidate for the treatment of refractory GISTs.