ABCB1 overexpression confers resistance to litronesib, an Eg5 inhibitor.

Eg5 (also known as kinesin family member 11, encoded by the KIF11 gene) is a promising mitotic target for cancer therapy, yet no Eg5 inhibitor has achieved FDA approval to date. Their limited clinical efficacy may be linked to multidrug resistance, potentially involving ATP-binding cassette (ABC) transporters. However, the extent and specificity of such interactions remain unclear. This study investigates the interaction of the clinically evaluated Eg5 inhibitor litronesib with major ABC transporters and its role in transporter-mediated resistance. Eg5 expression was analyzed using data from the TCGA and HPA datasets. High-throughput molecular docking was used to screen a panel of 25 representative Eg5 inhibitors against ABCB1, ABCC1, and ABCG2. Subsequent functional validation in paired parental and transporter-overexpressing cell lines involved cytotoxicity and reversal assays, a [3H]-paclitaxel accumulation assay, ATPase activity measurements, Western blotting, and immunofluorescence analysis. Database analysis revealed that Eg5 is overexpressed in multiple cancers and correlates with poor prognosis. High-throughput docking identified litronesib as a potential ABCB1 interactor with a strong predicted binding energy (-10.3 kcal/mol), while showing minimal predicted binding and no functional interaction with ABCC1 and ABCG2 in the models tested. ABCB1 overexpression conferred robust resistance to litronesib, which was reversed by verapamil. Litronesib induced a higher fold resistance than the classical ABCB1 substrates paclitaxel and doxorubicin (P < 0.05), and failed to reverse ABCB1-mediated multidrug resistance at non-cytotoxic concentrations. Mechanistically, litronesib strongly stimulated ABCB1 ATPase activity (EC50 ∼0.38 μM), modestly upregulated ABCB1 protein expression in a time-dependent manner, and enhanced intracellular [3H]-paclitaxel accumulation only at high concentrations, without altering ABCB1 subcellular localization. Litronesib exhibits pronounced ABCB1-mediated resistance, a finding that underscores the need for strategies such as patient stratification or structural optimization to enhance efficacy. These findings provide a crucial framework for the resistance-aware development of new Eg5 inhibitors and will inform their future clinical applications.