Identification of the protein-drug adduct formed between aldehyde dehydrogenase and S-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry

Disulfiram has been used clinically as an aversion therapy treatment for re covering alcoholics. One of its metabolites. S-metyl-N,N-diethylthiocarbamo yl sulfoside (MeDTC-SO), is currently believed by some to be the active met abolite in vivo. We demonstrate in this report that MeDTC-SO is a potent ir reversible inhibitor of I ecombinant rat liver mitochondrial aldehyde dehyd rogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde forme d during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC50 = 4.62 mu M. The inhibition of rlmAL DH was found to be accompanied by a concomitant increase of similar to 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spec trometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, me developed a no vel approach to characterize specific protein-drug interactions by Linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pe psin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH22 = 500.9, which was not present in the pepsin digestion of the non-inhibite d enzyme, This peptide was tentatively attributed to the putative active si te peptide (FNQGQC(301)C(302)C(303)) plus the adduct, This peptide was subj ected to analysis by LC/MS/MS, which allowed us to determine that the coval ent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme. Copyr ight (C) 2000 John Wiley & Sons, Ltd.