Disulfiram metabolites permanently inactivate the human multidrug resistance P-glycoprotein.
The human multidrug resistance P-glycoprotein (P-gp) uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. The relatively high expression of P-gp in organs such as the intestine, kidney, blood-brain/testes barrier and in some tumor cells can compromise chemotherapy treatments for patients with cancer or AIDS/HIV. It has been difficult to inhibit P-gp during chemotherapy with noncovalent inhibitors because the relatively high levels of inhibitors have severe side effects. An alternative approach to inhibit P-gp would be to covalently modify cysteine residues within the NBDs. In this study, we tested whether metabolites of disulfiram, a drug currently used to treat chronic alcoholism, could inhibit P-gp. We show that the disulfiram metabolites, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the verapamil-stimulated ATPase activity of P-gp with IC50 values (concentrations that result in 50% inhibition of activity) of 9 and 4.8 microM, respectively. Similarly, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the activity of aldehyde dehydrogenase with IC50 values of 3.2 and 1.7 microM, respectively. Inhibition of P-gp by the metabolites was not reversed by addition of the reducing compound, dithiothreitol. We then determined which endogenous cysteine residue was responsible for inhibiting P-gp activity after exposure to the disulfiram metabolites. Treatment of P-gp mutants containing a single cysteine residue showed that inactivation was primarily due to modification of Cys1074 in NBD2. These results indicate that metabolites of disulfiram can covalently inactivate P-gp. Covalent modification of drug transporters could be a useful approach for inhibiting their activities during chemotherapy.
Loo
Tip W
TW
CIHR Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Bartlett
M Claire
MC
Clarke
David M
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors; D018076:DNA, Complementary / Q000235:genetics; D004050:Ditiocarb / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009154:Mutation; D013329:Structure-Activity Relationship; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C504100:S-methyl N,N-diethylthiocarbamate sulfone; C073220:S-methyl N,N-diethylthiolcarbamate sulfoxide; D000255:Adenosine Triphosphate; D004050:Ditiocarb; D014700:Verapamil; D000444:Aldehyde Dehydrogenase; D000251:Adenosine Triphosphatases