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

Abstract

Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity. This live cell-based automated microscopy assay is very robust and allows simultaneous detection of cell permeable, non-toxic and potent inhibitors. The validity of the assay was demonstrated by profiling a library of 386 anti-cancer compounds, which are under clinical trials, for interactions with MRP1. The assay identified 12 potent inhibitors including two known MRP1 inhibitors, cyclosporine A and rapamycin. On the other hand, MRP1-inhibitory activity of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 has not been previously reported. Inhibition of MRP1 activity was further validated using flow cytometry and confocal microscopy for the respective detection of calcein and doxorubicin in MRP1-overexpressing cells. Among the identified compounds, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus resensitized MRP1-overexpressing H69AR cells towards vincristine, a cytotoxic chemotherapeutic agent, by 2-6-fold. Using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we also demonstrated that the identified compounds exert differential and selective response on the uptake of estradiol glucuronide, an endogenous MRP substrate. In summary, we demonstrated the effectiveness of the high content imaging-based high-throughput assay for profiling compound interaction with MRP1.

Small‐molecule inhibitors of multidrug resistance‐associated protein 1 and related processes: A historic approach and recent advances

“…Other kinase inhibitors, AZD1208, HS‐173, TAK‐733, and YM201636 (Figure ), were shown to inhibit the MRP1‐mediated calcein AM transport with IC 50 values of 0.7, 4.6, 8.5, and 2.0 μM . The compounds at 10 μM significantly increased the intracellular calcein concentration.…”

“…Other immunosuppressant agents have been evaluated in a screening by Peterson et al . Deforolimus (ridaforolimus), everolimus, sirolimus (rapamycin), and temsirolimus (torisel; Figure ) showed to inhibit the MRP1‐mediated calcein AM transport by IC 50 values of 4.9, 2.6, 2.8, and 5.6 μM, compared to cyclosporine A (6.6 μM) and MK571 (8.4 μM).…”

“…The authors found that this tricyclic dipiperidine derivative had an IC 50 of 12.4 μM, compared to the used standard inhibitor cyclosporine A (IC 50 = 8.8 μM). Another farnesyl protein transferase inhibitor, tipifarnib (Zarnestra; Figure ), was shown to inhibit the MRP1‐mediated calcein AM transport with an IC 50 value of 3.7 μM . This compound at 10 μM significantly increased the intracellular calcein concentration.…”

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA

“…CysLT1 activation contributes to various allergic reactions including bronchial constriction and asthma 47 . MK-571 is an orally active, potent and selective LTD4 antagonist (competes for CysLT1), and multidrug resistance protein 1 (MRP1) inhibitor 48 . While arachidonic acid by itself showed no improvements in transfection (Fig.…”

Multicellular tumor spheroids- an effectivein vitromodel for understanding drug resistance in head and neck cancer

“…We determined the median fluorescence intensity (MFI) values for the 2D cell cultures and MCSs obtained from LK0917, LK0902, and LK1108, in the presence and absence of the specific efflux pump inhibitors verapamil, novobiocin, and MK-571 against P-gp, BCRP, and MRP1, respectively, using flow cytometry analysis [37][38][39] . The MFI values for all transporters were comparable for both 2D cell cultures and MCSs as shown in Fig.…”