Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase
Introduction
1Antabuse (bis[diethylthiocarbamoyl] disulfide), also known as DSF, has been widely used clinically in the aversion therapy treatment of recovering alcoholics for over 50 years [1]. It is believed to be an irreversible inhibitor of mALDH, one of the key enzymes involved in alcohol metabolism [2]. Inhibition of ALDH leads to the accumulation of acetaldehyde after imbibing ethanol, and, thereby, induces a distinctly unpleasant DSF–ethanol reaction characterized by vasodilation, tachypnea, and tachycardia with subsequent nausea, vomiting, and hypotension [1]. DSF, the parent drug, is rapidly reduced in vivo to DDC [3]; however, it has been proposed that reoxidation of DDC to DSF is responsible for actual ALDH inhibition [4], [5], [6], [7], [8], [9], [10]. It has also been suggested that metabolites, such as MeDTC-SO and MeDTC-SO2, are responsible for the in vivo inactivation of the enzyme [11], [12], [13]. Previous studies have shown that MeDTC-SO is a potent, irreversible inhibitor of ALDH in vitro and in vivo[11], [13], [14]. In addition, this metabolite has been detected in rat plasma after DSF administration [11], [15], [16]. MeDTC-SO also inhibits recombinant human mALDH in vitro by forming a covalent adduct on one of the essential cysteines at the active site of the enzyme [17]. MeDTC-SO2 is also a potent inhibitor of ALDH in vitro[13], [14], yet, possibly due to its reactive nature, it has never been detected in vivo. In spite of the widespread use of this drug, the mechanism of inhibition of ALDH by DSF in vivo has not been determined to date.
Numerous strategies have been reported that purport to describe the interaction of a drug with its target protein(s). Such approaches typically include enzyme activity/inhibition assays, immunofluorescence, radioactive labeling, cross-linking, and protein binding assays, which all utilize nonspecific detection systems and in some cases require significant amounts of protein [18]. Furthermore, this information does not allow facile identification of the target protein, or determination of the mechanism of inhibition by the drug or its metabolite. More recently, electrospray ionization (or miniaturized versions such as microelectrospray and nanospray) in conjunction with mass spectrometry (ESI–MS) has played an increasingly important role in the rapid identification of proteins [19]. In particular, ESI–MS is now one of the preferred methods of choice in proteomic [20], functional proteomic [21], and functional genomic [22], [23] analyses. Furthermore, ESI–MS is proving to be highly complementary to multidimensional NMR. The former allows rapid structural determination of both native and modified proteins, requiring only femtomole–attomole amounts of analyte, while affording primary sequence information [19], [20].
In the present work, we describe a new, simple, but rapid, on-line sample processing and chromatographic separation method, used in conjunction with ESI–MS and ESI–tandem-MS (ESI–MS/MS) to structurally characterize in vivo derived protein–drug adducts, that overcomes the limitations of other generally used techniques (shown schematically in Fig. 1). We demonstrate the power of such an approach in determining both the site of adduct formation and the structure of the DSF metabolite–mALDH adduct produced after administering DSF to rats.
Section snippets
ALDH activity assay
The enzyme activity assay was performed as previously described [24], with some minor modifications. Briefly, 10 μL of purified mALDH was dispensed into the microtiter plate well in triplicates followed by the addition of 190 μL of 50 mM sodium pyrophosphate buffer, pH 8.8, and 25 μL of acetaldehyde and NAD+. The final acetaldehyde concentration was 160 μM, and that of NAD+ was 500 μM. Enzyme activity was determined, in part, by spectrophotometrically monitoring NADH formation at 340 nm using a
Results
DSF has been used as an aversion therapy treatment for recovering alcoholics for over five decades [1]. However, the in vivo mechanism of inhibition still has not been directly determined. Hence, we were interested in discovering the precise mechanisms and site of inhibition of mALDH in vivo.
Discussion
We have shown in this report that DSF is a potent active site inhibitor in vivo in rats. Numerous contrary hypotheses have been made in the past with regard to the mode of interaction between ALDH and DSF in vivo. It is believed by some that DSF inhibits ALDH by forming a mixed disulfide at the active site [4], [6], [38], while others report that the inhibition may be due to formation of an intramolecular disulfide bridge [33], [39]. In addition, it has been reported that DSF inhibits horse
Acknowledgments
This research was supported by NIH Grant AA09543 (J.J.L.) and Finnigan MAT No. 1 (S.N.). The authors thank Mrs. Diana Ayerhart for her assistance in preparing this manuscript and Dr. W. Stephen Brimijoin for his assistance in administering DSF to the animals.
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