Ensartinib (X-396) is a promising tyrosine kinase inhibitor currently undergoing advanced clinical evaluation for the treatment of non-small cell lung cancer. In this work, we investigate possible interactions of this promising drug candidate with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs), which play major roles in multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). Accumulation studies showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 transporters. Additionally, incubation experiments with recombinant CYPs showed that ensartinib significantly inhibits CYP3A4 and CYP2C9. Subsequent molecular docking studies confirmed these findings. Drug combination experiments demonstrated that ensartinib synergistically potentiates the antiproliferative effects of daunorubicin, mitoxantrone, and docetaxel in ABCB1, ABCG2, and CYP3A4-overexpressing cellular models, respectively. Advantageously, ensartinib's antitumor efficiency was not compromised by the presence of MDR-associated ABC transporters, although it acted as a substrate of ABCB1 in Madin-Darby Canine Kidney II (MDCKII) monolayer transport assays. Finally, we demonstrated that ensartinib had no significant effect on the mRNA-level expression of examined transporters and enzymes in physiological and lung tumor cellular models. In conclusion, ensartinib may perpetrate clinically relevant pharmacokinetic DDIs and modulate ABCB1-, ABCG2-, and CYP3A4-mediated MDR. The in vitro findings presented here will provide a valuable foundation for future in vivo investigations.

Department of Biochemical Sciences Faculty of Pharmacy in Hradec Kralove Charles University Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

Department of Chemistry Faculty of Science University of Hradec Králové Hradecká 1285 Hradec Králové 500 03 Czech Republic

Department of Pharmacology and Toxicology Faculty of Pharmacy in Hradec Kralove Charles University Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

Division of Biochemical Toxicology National Center for Toxicological Research U S FDA 3900 NCTR Road Jefferson AR 72079 USA

Institute of Biotechnology Brandenburg University of Technology Cottbus Senftenberg Universitätsplatz 1 01968 Senftenberg Germany

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