Inhibition of recombinant human mitochondrial and cytosolic aldehyde dehydrogenases by two candidates for the active metabolites of disulfiram.
We expressed recombinant human cytosolic (ALDH1, high Km) and mitochondrial aldehyde dehydrogenase (ALDH2, low Km) in Escherichia coli and purified the enzymes to homogeneity to examine the nature of inhibition of human ALDH by disulfiram, its confirmed metabolite S-methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide, and its proposed metabolite MeDTC sulfone. Disulfiram, MeDTC sulfoxide, and MeDTC sulfone, respectively, were potent inhibitors with IC50 values of 0.15 +/- 0.02 microM, 0.27 +/- 0.04 microM, and 0.12 +/- 0.02 microM for ALDH1, and 1.45 +/- 0.40 microM, 1.16 +/- 0.56, and 0.40 +/- 0.10 microM for ALDH2. Extensive dialysis did not restore the activity of the inactivated enzyme, indicating irreversible inhibition. Both the esterase and dehydrogenase activities of ALDH2 were inhibited to the same extent by MeDTC sulfone and sulfoxide, suggesting that both catalytic sites are closely linked. The time course of inhibition of ALDH appeared to be first-order for both MeDTC sulfone and MeDTC sulfoxide. Kitz and Wilson plots of the half-life of inactivation versus 1/[inhibitor] indicated that the reactions between ALDH and inhibitors were bimolecular. The pseudobimolecular rate constants (k3/KI) for the ALDH-inhibitor reactions were 1 x 10(5), 1 x 10(4), 3 x 10(3), and 1 x 10(3) s-1 M-1 ALDH1-sulfone, ALDH1-sulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide, respectively. ALDH2 was not significantly protected from inactivation from either MeDTC sulfoxide or MeDTC sulfone by NAD alone, but high concentrations of NAD and acetaldehyde completely prevented inhibition. Since disulfiram is rapidly metabolized in vivo, it is believed that disulfiram is too short-lived to inhibit ALDH directly. The results of our study indicate that MeDTC sulfoxide and sulfone are potent inhibitors of human ALDH and are reasonable candidates for the proximal inhibitors of ALDH following disulfiram administration.
Lam
J P
JP
Clinical Pharmacology Unit, Department of Pharmacology, Mayo Medical School, Rochester, Minnesota 55905, USA.
Mays
D C
DC
Lipsky
J J
JJ
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D004221:Disulfiram / Q000378:metabolism / Q000494:pharmacology*; D004050:Ditiocarb / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors*; D013450:Sulfones / Q000494:pharmacology
D007527:Isoenzymes; D011994:Recombinant Proteins; D013450:Sulfones; C073220:S-methyl N,N-diethylthiolcarbamate sulfoxide; D004050:Ditiocarb; D000444:Aldehyde Dehydrogenase; D004221:Disulfiram