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

Abstract

Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.

Disulfiram Metabolites Permanently Inactivate the Human Multidrug Resistance P-Glycoprotein

“…Inactivation of ADH during aversion therapy in chronic alcoholism likely involves carbamoylation of an essential cysteine. Treatment of rat mitochondrial ADH in vitro with DETC-sulfoxide followed by proteolysis and mass spectrometry showed that inhibition of activity was due to carbamoylation of Cys302. , Similar findings were reported when mitochondrial ADH was isolated from rats that had been administered disulfiram. , Like ADH, we found that inhibition of P-gp with either DETC-sulfoxide or DETC-sulfone was mainly due to modification of one particular cysteine, Cys1074 in NBD2 (Figure ). It has been shown that modification of only one of the Walker A cysteines with N -ethylmaleimide 16,48 or with disulfiram 20 was sufficient to inactivate P-gp.…”

“…Treatment of rat mitochondrial ADH in vitro with DETC-sulfoxide followed by proteolysis and mass spectrometry showed that inhibition of activity was due to carbamoylation of Cys302. 35,46 Similar findings were reported when mitochondrial ADH was isolated from rats that had been administered disulfiram. 19,47 Like ADH, we found that inhibition of P-gp with either DETC-sulfoxide or DETCsulfone was mainly due to modification of one particular cysteine, Cys1074 in NBD2 (Figure 6).…”

Zinc diethyldithiocarbamate allergenicity: potential haptenation mechanisms

“…Hepatic metabolism of TETD has been reported in the literature as it is used clinically (as disulfiram) for the treatment of alcoholism (14, 26–31). Oxidative desulfuration, similar to the direct oxidation performed with ZDEC in this study, was observed with rat and human liver microsomes in the presence of reduced nicotinamide adenine dinucleotide phosphate (26).…”

Development of an automated on-line pepsin digestion–liquid chromatography–tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents

“…Due to the long manual sample handling steps, these methods are time-consuming and rather laborious. Automated on-line systems, as reported earlier by Shen et al [19] and more recently by Hoos et al [20], will afford increased sample throughput after terrorist or military incidents and will also be advantageous for use in field laboratories. We here report the development of an automated configuration to verify and monitor CWA (chemical warfare agent) exposures, using on-line enzymatic digestion-LC-tandem MS.…”