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.