Identification of an N-methylpyrazole derivative as a selective inhibitor of breast cancer resistance protein (BCRP/ABCG2)

Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters remains a major obstacle to cancer chemotherapy, particularly at later disease stages with metastases. Among the 48 human ABC proteins, P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are the most studied ABC transporters associated with MDR. Inhibition of ABC transporters has been considered as one possible strategy to overcome MDR. In this study, twelve N-methylpyrazole derivatives were evaluated as inhibitors of ABCB1, ABCC1, and ABCG2. Several compounds selectively inhibited ABCG2 while showing no activity against ABCB1 or ABCC1. The most potent derivative, 1l , inhibited more than 50% of ABCG2 activity at 10 μM and displayed substrate-independent inhibition, with IC 50 values ranging from 1.6 to 3.8 μM depending on the fluorescent probe used. Compound 1l exhibited only mild cytotoxicity and was not transported itself by ABCG2. Mechanistic studies revealed that 1l induced conformational changes in ABCG2, as evidenced by increased binding of the 5D3 conformational antibody. Combination assays with established ABCG2 inhibitors, including chromone 4a and indeno-[1,2-b]indole 5e , showed neither synergistic nor antagonistic effects. Induced-fit docking simulations supported the experimental data, indicating that 1l binds within the central transmembrane cavity of ABCG2, engaging key residues such as Phe439 and stabilizing inward-facing conformations. Finally, co-treatment with 1l restored sensitivity of ABCG2-overexpressing cells to the anticancer drug SN38, effectively reversing the MDR phenotype. Collectively, these results identify N-methylpyrazole derivatives as promising selective inhibitors of ABCG2. • N -methylpyrazoles act as selective inhibitors of ABCG2 over P-gp and MRP1. • Compound 1l shows substrate-independent ABCG2 inhibition with low cytotoxicity. • Compound 1l restores SN38 sensitivity in ABCG2-overexpressing resistant cells.