Befotertinib restored chemosensitivity to multidrug-resistant cancer cells by inhibiting the drug efflux activity of ABCB1

Objectives Chemotherapy remains central to cancer management, but its effectiveness is often limited by multidrug resistance (MDR), largely mediated by the ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein; P-gp). As no clinically approved inhibitors are available to overcome ABCB1-mediated MDR, this study aimed to evaluate befotertinib (D-0316), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved by the National Medical Products Administration, for its potential to modulate ABCB1 activity. Methods Cytotoxicity and apoptosis assays were used to assess chemosensitization in multidrug-resistant cancer cells. Drug efflux inhibition was examined, while vanadate-sensitive ATPase activity and molecular docking analyses were performed to characterize interactions between befotertinib and ABCB1. Key findings Befotertinib significantly enhanced anticancer drug-induced apoptosis and resensitized resistant cells in a concentration-dependent manner. It inhibited ABCB1-mediated efflux, stimulating ATPase activity and demonstrating direct binding to key residues within the ABCB1 substrate-binding pocket. Conclusions These results identify befotertinib as a functional inhibitor of ABCB1 with the capacity to reverse MDR. The findings support its potential as a repurposed agent for combination therapy in tumors with high ABCB1 expression, offering a practical strategy to enhance chemotherapy efficacy.