High-content screening of clinically tested anticancer drugs identifies novel inhibitors of human MRP1 (ABCC1)

Brian G Peterson 1, Kee W Tan 1, Bremansu Osa-Andrews 1, Surtaj H Iram 2

Abstract
Multidrug resistance protein 1 (MRP1/ABCC1) is an integral membrane efflux transporter and a member of the ATP-binding cassette (ABC) protein superfamily. MRP1 plays a key role in the transport and distribution of a broad range of endogenous compounds and xenobiotics, including many therapeutic drugs, across various tissues and physiological barriers. Overexpression of MRP1 has been strongly linked to multidrug resistance in cancer chemotherapy.

In this study, we developed a robust, high-content imaging-based screening assay to assess MRP1 activity in live cells. This automated microscopy assay enables the simultaneous identification of potent, cell-permeable, and non-toxic MRP1 inhibitors. To validate the assay, we screened a library of 386 anticancer compounds currently in clinical trials for their interaction with MRP1. The assay identified 12 potent inhibitors, including two well-known MRP1 inhibitors—cyclosporine A and rapamycin. Notably, the MRP1-inhibitory effects of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 had not been previously reported.

Inhibition of MRP1 was further confirmed using flow cytometry and confocal microscopy to detect intracellular accumulation of calcein and doxorubicin in MRP1-overexpressing cells. Among the newly identified inhibitors, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus sensitized MRP1-overexpressing H69AR cells to vincristine by 2- to 6-fold. Additionally, using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we demonstrated that these compounds showed selective and differential inhibition of estradiol glucuronide uptake, a known MRP substrate.

In summary, our study highlights the utility of a high-content imaging-based, HS-173 high-throughput assay for profiling compound interactions with MRP1, offering a valuable tool for identifying novel MRP1 inhibitors and overcoming drug resistance in cancer.