In vivo inhibition of aldehyde dehydrogenase by disulfiram |
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| Affiliation: | 1. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN 55905, USA;2. Clinical Pharmacology Unit, Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN 55905, USA;3. Department of Internal Medicine, Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN 55905, USA;4. Biomedical Mass Spectrometry and Functional Proteomics Facility, Guggenheim C-009B, Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN 55905, USA;5. Department of Biochemistry and Molecular Biology and Division of Biomedical Engineering, Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN 55905, USA;1. INQUINOA-CONICET, Instituto de Química Física, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, San Lorenzo 456, T4000CAN, Tucumán, Argentina;2. CEQUINOR, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C. C. 962, 1900 La Plata, Argentina;3. Departamento de Ciencias Básicas, Universidad Nacional de Luján, Ruta 5 y 7, 6700 Luján, Buenos Aires, Argentina;1. Instituto de Investigaciones en Fisicoquímica de Córdoba (INFIQC, CONICET), Dpto. Química Orgánica, Fac. Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, X5000HUA, Argentina;2. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica (UNITEFA, CONICET), Dpto. Farmacia, Fac. Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, X5000HUA, Argentina;1. Institute of Medical Microbiology, University of Zurich, Gloriastrasse 30/32, CH-8006 Zurich, Switzerland;1. Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan;2. Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan;1. Hematology Department, School of Allied Medicine, Tehran University of Medical Sciences, Tehran, Iran;2. Hematologic Malignancies Research Center, Tehran University of Medical Sciences, Tehran, Iran |
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| Abstract: | Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein–drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of ∼71 and ∼100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH+=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC301C302C303, derived from the enzyme active site region, and were modified at Cys302 by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). |
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