Overview - In vitro inhibition of aldehyde dehydrogenase by disulfiram andmetabolites

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 i nhibition is still not known. In this work we describe the in vitro interac tions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocar bamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directl y inhibits rmALDH (IC50 = 36.4 muM) by inducing the formation of an intramo lecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge forme d involves two of the three cysteines located at the active site of the enz yme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that th e electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC50 = 4.62 muM). We isolate and identify a carbamoylated peptide at Cys(302) with the seque nce FNQGQC(301)C(302)C(303). Hence we show that MeDTC-SO exhibits its inhib itory effect by covalently modifying the -SH side-chain of Cys(302), presen t 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 inhib it the enzyme by acting directly at the active site of specific monomers of rmALDH. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.