MECHANISMS AND CHEMISTRY OF DEHYDROGENASE INACTIVATORS

羟基类固醇脱氢酶(HSD)是S的重要组成部分 类固醇代谢酶的一类。可逆性 由这些酶介导的氧化还原酶反应 对皮质类固醇激素的荷尔蒙活动有显著影响， 孕激素、雄激素和雌激素。通过使用基于机制的 HSD不可逆抑制剂及其作用机制的研究 失活，与药理相关的信息 可以获得对这类酶的操纵。 这项建议的具体目标是： 1)新型机理不可逆缓蚀剂的合成 三个不同的HSD， 2)这些抑制剂的酶学评价，以及 3)共价态HSD-抑制剂的结构解析 加合物。 所提出的基于机理的抑制剂是乙烯醇。 其被特定HSD的酶催化氧化将产生 可被酶攻击的共轭乙酰基酮 亲核剂形成共价加合物，从而使 酵素。三种不同系列的乙炔化合物，一种 三种羟基类固醇脱氢酶系列 包括在研究中，将做好准备。运动学和机械学 结构/活动关联所需的数据为 从测量HSD在以下条件下的失活率获得 各种实验条件。结构数据将是 从碳-13核磁推论(核磁共振)中获得 用酶产生的HSD灭活的实验 含碳-13低于99%的炔基酮 在乙酰键的两个碳原子中都有富集物。解 和/或固态核磁共振实验。结构性 基于这些碳-13化学位移数据得出的结论 核磁共振实验将通过比较 观察到的化学位移与适当模型中发现的化学位移 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.