Doxorubicin as a fluorescent reporter identifies novel MRP1 (ABCC1) inhibitors missed by calcein-based high content screening of anticancer agents

Abstract

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum multidrug resistance and can decrease the bioavailability of many important drugs. Substrates of MRP1 include anti-cancer agents, antibiotics, antivirals, antidepressants and anti-inflammatory drugs. Using calcein as a fluorescent reporter in a high content uptake assay, we recently reported the identification of 12 MRP1 inhibitors after screening an anti-cancer library of 386 compounds. Here, we describe the development of a new high content imaging-based uptake assay using doxorubicin as a fluorescent reporter. Screening the same anti-cancer library of 386 compounds, the new assay identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. Inhibition of MRP1 activity was confirmed using flow cytometry and confocal microscopy-based transport assays. Six drugs (afatinib, celecoxib, doramapimod, mifepristone, MK-2206 and rosiglitazone) were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against vincristine, doxorubicin and etoposide. Mifepristone and doramapimod were most effective in reversal of resistance against vincristine while mifepristone and rosiglitazone were most successful in resensitizing H69AR cells against doxorubicin. Furthermore, resistance towards etoposide was completely reversed in the presence of celecoxib or doramapimod. Selected drugs were also evaluated for resistance reversal in HEK cells that overexpress P-glycoprotein or breast cancer resistance protein. Our results indicate mifepristone and doramapimod as pan inhibitors of these three drug transporters while celecoxib exhibited selective MRP1 inhibition. Together, our findings signify the importance of MRP1 in drug discovery and demonstrate the effectiveness and value of doxorubicin-based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies. On the other hand, anti-cancer drugs that did not interact with MRP1 carry a low risk for developing MRP1-mediated resistance.