title: In vivo inhibition of aldehyde dehydrogenase by disulfiram.
abstract: Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+)=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).
authors: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic/Foundation, 200 First Street SW, 55905, Rochester, MN, USA.
authors: J J
authors: JJ
authors: Lipsky
authors: M L
authors: ML
authors: Shen
authors: S
authors: S
authors: Naylor
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004221:Disulfiram / Q000378:metabolism / Q000494:pharmacology*; D004050:Ditiocarb / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology; D010434:Pepsin A; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004791:Enzyme Inhibitors; C073220:S-methyl N,N-diethylthiolcarbamate sulfoxide; D004050:Ditiocarb; D000444:Aldehyde Dehydrogenase; D010434:Pepsin A; D004221:Disulfiram