MECHANISMS AND CHEMISTRY OF DEHYDROGENASE INACTIVATORS

羟基类固醇脱氢酶（HSD）是一种重要的 一类类固醇代谢酶。 可逆 由这些酶介导的氧化还原酶反应 对皮质类固醇激素活性的显著影响， 孕激素、雄激素和雌激素。 通过使用基于机制的 不可逆的抑制剂，并研究其作用机制， 灭活，药理学相关信息 可以获得这类酶的操作。 这项建议的具体目标是： 1)基于机理新型不可逆缓蚀剂的合成 三种不同的高速公路系统， 2)这些抑制剂的酶促评价，以及 3)共价键合的HSD-inhibitor的结构解析 加合物 提出的基于机理的抑制剂是炔醇 其通过特定HSD的酶促氧化将产生 能被酶攻击的共轭炔酮 亲核试剂以形成共价加合物，从而使 酵素 三种不同系列的炔属化合物，一种 三种羟基类固醇脱氢酶中每一种的系列 研究报告中，将提出。 动力学和机械学 结构/活性相关性所需的数据将是 从HSD失活率的测量中获得， 各种实验条件。 结构数据将 由碳-13核磁共振（NMR）获得 用酶产生的HSD灭活的实验 碳-13含量低于99%的炔酮 在炔键的两个碳原子中富集。 溶液 和/或固态NMR实验。 结构 根据这些碳-13化学位移数据得出的结论 NMR实验将通过比较 观察到的化学位移与适当模型中发现的化学位移一致 加合物

Hydroxysteroid dehydrogenases (HSD)s constitute an important class of steroid metabolizing enzymes. The reversible oxidoreductase reactions mediated by these enzymes have dramatic effects on the hormonal activities of corticosteroids, progestins, androgens, and estrogens. By using mechanism-based irreversible inhibitors of HSDs and studying their mechanisms of inactivation, information relevant to the pharmacologic manipulation of this class of enzymes can be obtained. The specific aims of this proposal are: 1) the synthesis of novel mechanism-based irreversible inhibitors of three different HSDs, 2) the enzymatic evaluation of these inhibitors, and 3) the elucidation of the structures of covalent HSD-inhibitor adducts. The proposed mechanism-based inhibitors are acetylenic alcohols whose enzymatic oxidation by a particular HSD will generate a conjugated acetylenic ketone that can be attacked by an enzyme nucleophile to form a covalent adduct and thus inactivate the enzyme. Three different series of acetylenic compounds, one series for each of the three hydroxysteroid dehydrogenases included in the study, will be prepared. Kinetic and mechanistic data needed for the structure/activity correlations will be obtained from measurements of rates of HSD inactivation under a variety of experimental conditions. Structural data will be obtained from carbon-13 nuclear magnetic reasonance (NMR) experiments done with HSDs inactivated by enzyme-generated acetylenic ketones that contain less than 99% carbon-13 enrichment in both carbon atoms of the acetylenic bond. Solution and/or solid state NMR experiments are proposed. Structural conclusions based on carbon-13 chemical shift data from these NMR experiments will be made possible by comparison of the observed chemical shifts with those found in appropriate model adducts.