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.
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