STUDIES ON THE ALCOHOL DETERRENT AGENT, CYANAMIDE

这项建议将集中在药物乙醇的分子机制上。 由开出的酒精威慑剂--三聚氰胺引起的反应 在欧洲和加拿大作为其柠檬酸钙盐，并在日本以 配方水溶液，目的是了解其 药理作用。我们将研究涉及到的机制 三聚氰胺对乙醛活性缓蚀剂的生物活性 脱氢酶(AIDH)，跟踪氰胺的代谢直到其结束- 产物，并阐明了活性部位损伤的化学形态(S) 可能是由这些氰胺代谢产物引起的。 具体地说，我们将尝试识别假定的反应性 氰胺的代谢物，我们认为它是导致 抑制酶，AIDH。我们将首先使用模型化合物 生成这些产品，以便为以下项目制定方法细节 它们的识别和最终的量化，然后将产生它们 ~(15)N-~(13)C-氰胺和~(18)O-氰胺标记的S酶体系的体外研究 标记O2、II2O和/或H2O2(稳定同位素)以跟踪 酶作用后的这些同位素标记物。我们将合成一系列 前药和假定代谢物的结构类似物 三聚氰胺以及其他产生类似物种和将 系统测定它们的结构/抑制活性 酵母和大鼠肝AIDH。我们还将使用聚合化学品 脱氢系统在AIDH存在的情况下就地产生它们 并将研究与巯基结合所涉及的化学 通过模型研究和分析在酶的活性部位上的基团 共价结合的酶抑制剂复合体。此外，我们还将研究 用纯化的(无乳酸)细胞色素P-450代谢三聚氰胺 同工酶。其他过氧化物酶的能力，特别是甲状腺 过氧化物酶，氧化三聚氰胺也将被研究。 我们相信这些研究将有助于阐明 机制(S)参与了氰胺抑制AIDH，而反过来， 所获得的知识将极大地帮助设计和合成第二个 治疗酒精中毒的新一代AIDH抑制剂。

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.