Overview--in vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites.
Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC(50)=36.4 microM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC(50)=4.62 microM). We isolate and identify a carbamoylated peptide at Cys(302) with the sequence FNQGQC(301)C(302)C(303). Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys(302), present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.
Lipsky
J J
JJ
Department of Molecular Pharmacology and Experimental Therapeutics, Guggenheim, Mayo Clinic/Foundation, 200 First Street, SW, 55905, Rochester, MN, USA.
Shen
M L
ML
Naylor
S
S
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004221:Disulfiram / Q000378:metabolism / Q000494:pharmacology*; D004050:Ditiocarb / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C073220:S-methyl N,N-diethylthiolcarbamate sulfoxide; D004050:Ditiocarb; D000444:Aldehyde Dehydrogenase; D003545:Cysteine; D004221:Disulfiram