MECHANISM OF ENZYME INHIBITION BY PHARMACOLOGICAL AGENTS

描述：哺乳动物磷酸二酯酶(PDE)水解环状 核苷酸，在cAMP介导的信号调节中是必不可少的 转导。它们构成了一大组同工酶，可以 分成家族或类型的。显示不同的PDE类型 独特的组织分布，独特的生化特性和 独特的抑制剂配置文件。这些特征解释了生理上的 这些酶的性质，并与PDE的疗效直接相关 作为治疗剂的抑制剂。不同PDE类型的序列比对 显示了一些守恒性，可能反映了对催化剂的要求 活动。负责的特定区域和特定残留物 然而，每种PDE类型的独特性质尚不清楚。 我们之前设计了一种酵母表达系统来分析哺乳动物 用于这些酶的抗药性突变体的遗传选择。 使用缺失和点突变来识别区域和残基 活性和药物结合所必需的。我们现在建议研究一下 利用一种改进的方法研究几种PDE类型的同工酶特异性 这建立在我们重组酵母系统的优势之上。我们会 利用嵌合的PDE酶来确定哪些残基是足够的， 在PDE的背景下，授予独特的生化特性和 每种PDE类型的抑制剂敏感性。这些数据将用于 优化抗药性突变体的选择，从而导致 鉴定说明这些特性的特定残基。 还提出了检测底物专一性的实验。结果是 将解决同工酶特异性决定因素是否定位于 在不同形式的偏微分方程组中保守的模式。此外， 从这些研究中获得的信息应该对以PDE为中心的 治疗学，同工酶之间的区别是至关重要的。它还可以服务于 作为分析其他复杂同工酶家族的模型。

DESCRIPTION: Mammalian phosphodiesterases (PDEs) hydrolyze cyclic nucleotides and are essential in the regulation of cAMP-mediated signal transduction. They constitute a large group of isozymes that can be categorized into families or types. Different PDE types display characteristic tissue distributions, distinctive biochemical properties and unique inhibitor profiles. These features account for the physiological properties of these enzymes and are directly relevant to the efficacy of PDE inhibitors as therapeutic agents. Sequence alignment of different PDE types shows some conservation that is likely to reflect requirements for catalytic activity. The specific regions, and the particular residues, responsible for the distinctive properties of each PDE type are not yet known, however. We previously devised a yeast expression system for analysis of mammalian PDEs and for genetic selection of drug resistant mutants of these enzymes. Deletions and point mutations were used to identify regions and residues necessary for activity and for drug binding. We now propose to examine isozyme specificity in several PDE types, making use of a modified approach that builds on the strengths of our reconstituted yeast system. We will utilize chimeric PDE enzymes to determine which residues are sufficient, within the context of a PDE, to confer the unique biochemical properties and inhibitor susceptibility of each PDE type. These data will be used to optimize the selection of drug-resistant mutants leading to the identification of specific residues that account for these properties. Experiments to examine substrate specificity are also proposed. The results will address whether isozyme specificity determinants are localized in a pattern that is conserved across divergent forms of PDEs. In addition, the information derived from these studies should be useful for PDE-centered therapeutics where distinction among isozymes is crucial. It may also serve as a model for the analysis of other complex isozyme families.