The Molecular Basis of the Action of Disulfiram as a Modulator of the Multidrug Resistance-Linked ATP Binding Cassette Transporters MDR1 (ABCB1) and MRP1 (ABCC1)

Abstract

The overexpression of multidrug resistance protein 1 (MDR1) and multidrug resistance protein 1 (MRP1) gene products is a major cause of multidrug resistance in cancer cells. A recent study suggested that disulfiram, a drug used to treat alcoholism, might act as a modulator of P-glycoprotein. In this study, we investigated the molecular and chemical basis of disulfiram as a multidrug resistance modulator. We demonstrate that in intact cells, disulfiram reverses either MDR1-or MRP1-mediated efflux of fluorescent drug substrates. Disulfiram inhibits ATP hydrolysis and the binding of [␣-32 P]8-azidoATP to Pglycoprotein and MRP1, with inhibition curves comparable with those of N-ethylmaleimide, a cysteine-modifying agent. However, if the ATP sites are protected with excess ATP, disulfiram stimulates ATP hydrolysis by both transporters in a concentration-dependent manner. Thus, in addition to modifying cysteines at the ATP sites, disulfiram may interact with the drugsubstrate binding site. We demonstrate that disulfiram, but not N-ethylmaleimide, inhibits in a concentration-dependent manner the photoaffinity labeling of the multidrug transporter with 125 I-iodoarylazidoprazosin and [3 H]azidopine. This suggests that the interaction of disulfiram with the drug-binding site is independent of its role as a cysteine-modifying agent. Finally, we have exploited MRP4 (ABCC4) to demonstrate that disulfiram can inhibit ATP binding by forming disulfide bonds between cysteines located in the vicinity of, although not in, the active site. Taken together, our results suggest that disulfiram has unique molecular interactions with both the ATP and/or drug-substrate binding sites of multiple ATP binding cassette transporters, which are associated with drug resistance, and it is potentially an attractive agent to combat multidrug resistance.

The Cancer Stem Cell Marker Aldehyde Dehydrogenase Is Required to Maintain a Drug-Tolerant Tumor Cell Subpopulation

“…Other reported mechanisms of action of disulfiram, including inhibition of P-glycoproteins involved in drug efflux and proteasome inhibition, are seen at relatively higher disulfiram concentrations (37,38). However, our findings indicating complete inhibition of phospho-MET in MKN-45 and GTL-16 DTPs (Supplementary Fig.…”

Modulation of the function of the multidrug resistance–linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin

“…2C, during 60-minute incubation, curcumin I is not metabolized, indicating that the curcumin I itself interferes with the binding of the transport substrate. Taken together, the transport and the photolabeling assay clearly showed that curcuminoids inhibit the binding of the substrates to the transporter and prevent efflux of We reported earlier that that compounds, such as disulfiram, interact with both the ATP-binding site and the substrate-binding site of ABCB1 (36). Therefore, the effect of curcuminoids on the binding of [a-…”

“…Photoaffinity ]azidopine into the ABCG2 band was quantified by estimating the radioactivity incorporated into the band using the STORM 860 PhosphorImager System (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuant as described (36). 6 cells.…”

Sunitinib (Sutent, SU11248), a Small-Molecule Receptor Tyrosine Kinase Inhibitor, Blocks Function of the ATP-Binding Cassette (ABC) Transporters P-Glycoprotein (ABCB1) and ABCG2

“…Samples were separated on a 7% Tris-acetate gel at constant voltage, and gels were dried and exposed to X-ray film for 12 to 24 h at Ϫ80°C. The incorporation of [ 125 I]IAAP into the ABCG2 or P-gp band was quantified using the STORM 860 PhosphorImager system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and the software ImageQuaNT (GE Healthcare), as described previously (Sauna et al, 2004).…”