This proposal will focus on the molecular mechanism of the drug-ethanol reaction (DER)elicited by cyanamide, an alcohol deterrent agent prescribed in Europe and Canada as its citrated calcium salt and available in Japan as formulated aqueous solutions, with the aim of understanding its pharmacological action. We will study the mechanisms involved in the bioactivation of cyanamide to the active inhibitor of aldehyde dehydrogenase (AIDH), trace the metabolism of cyanamide to its end- products, and elucidate the chemical form(s) of the active-site lesion that might be elicited by these cyanamide metabolites. Specifically, we will attempt to identify the putative reactive metabolites of cyanamide which we believe to be responsible for the inhibition of the enzyme, AIDH. We will first use model compounds that generate these products in order to work out the methodological details for their identification and eventual quantitation, then will produce them with enzyme system(s) in vitro using labeled 15N- & 13C-cyanamide and 18O- labeled O2, II2O, and/or H2O2 (stable isotopes) to trace the course of these isotopic labels after enzymatic action. We will synthesize a series of prodrugs and structural analogs of the postulated metabolite of cyanamide as well as other prodrugs that generate like species and will systematically determine their structure/inhibitory activities against yeast and rat liver AIDH. We will also use polymeric chemical dehydrogenation systems to generate them in situ in the presence of AIDH and will investigate the chemistry involved in binding to the sulfhydryl group at the active-site of the enzyme by model studies and by analysis of the covalently-bound enzyme inhibitor complex. In addition, we will study the metabolism of cyanamide using purified (acatalactic) cytochrome P-450 isozymes. The ability of other peroxidase enzymes, in particular, thyroid peroxidase, to oxidize cyanamide will also be studied. We believe that these studies will help elucidate the molecular mechanism(s) involved in AIDH inhibition by cyanamide, and, in turn, the knowledge gained will greatly aid in the design and synthesis of second generation AIDH inhibitors for the treatment of alcoholism.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Research Project (R01)
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Biochemistry, Physiology and Medicine Subcommittee (ALCB)
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University of Minnesota Twin Cities
Schools of Pharmacy
United States
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DeMaster, E G; Redfern, B; Nagasawa, H T (1998) Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide. Biochem Pharmacol 55:2007-15
Conway, T T; DeMaster, E G; Lee, M J et al. (1998) Prodrugs of nitroxyl and nitrosobenzene as cascade latentiated inhibitors of aldehyde dehydrogenase. J Med Chem 41:2903-9
Shirota, F N; Stevens-Johnk, J M; DeMaster, E G et al. (1997) Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo. J Med Chem 40:1870-5
Shirota, F N; Goon, D J; DeMaster, E G et al. (1996) Nitrosyl cyanide, a putative metabolic oxidation product of the alcohol-deterrent agent cyanamide. Biochem Pharmacol 52:141-7
Nagasawa, H T; DeMaster, E G; Goon, D J et al. (1995) Carbethoxylating agents as inhibitors of aldehyde dehydrogenase. J Med Chem 38:1872-6
Nagasawa, H T; Kawle, S P; Elberling, J A et al. (1995) Prodrugs of nitroxyl as potential aldehyde dehydrogenase inhibitors vis-a-vis vascular smooth muscle relaxants. J Med Chem 38:1865-71
Fukuto, J M; Gulati, P; Nagasawa, H T (1994) Involvement of nitroxyl (HNO) in the cyanamide-induced vasorelaxation of rabbit aorta. Biochem Pharmacol 47:922-4
Nagasawa, H T; Yost, Y; Elberling, J A et al. (1993) Nitroxyl analogs as inhibitors of aldehyde dehydrogenase. C-nitroso compounds. Biochem Pharmacol 45:2129-34
Fukuto, J M; Hszieh, R; Gulati, P et al. (1992) N,O-diacylated-N-hydroxyarylsulfonamides: nitroxyl precursors with potent smooth muscle relaxant properties. Biochem Biophys Res Commun 187:1367-73
Lee, M J; Elberling, J A; Nagasawa, H T (1992) N1-hydroxylated derivatives of chlorpropamide and its analogs as inhibitors of aldehyde dehydrogenase in vivo. J Med Chem 35:3641-7

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