MECHANISM OF ENZYME INHIBITION BY PHARMACOLOGICAL AGENTS

药理制剂抑制酶的机制

• 批准号: 2714522
• 负责人: JOHN J. COLICELLI
• 金额: $ 10.44万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2714522
• 关键词: active sites; chimeric proteins; drug resistance; enzyme activity; enzyme complex; enzyme mechanism; enzyme substrate; gene deletion mutation; isozymes; mutant; phosphodiesterase inhibitors; phosphodiesterases; point mutation; stress proteins; tissue /cell culture; western blottings; yeasts

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.

描述：哺乳动物磷酸二酯酶 (PDE) 水解环状化合物 核苷酸，对于调节 cAMP 介导的信号至关重要 转导。 它们构成了一大组同工酶，可以 分为家族或类型。 显示不同的 PDE 类型 特征性组织分布、独特的生化特性和 独特的抑制剂概况。 这些特征说明了生理学 这些酶的特性与 PDE 的功效直接相关 抑制剂作为治疗剂。 不同 PDE 类型的序列比对 显示出一些可能反映催化要求的守恒 活动。 负责的特定区域和特定残基 然而，每种偏微分方程的独特性质尚不清楚。 我们之前设计了一种用于分析哺乳动物的酵母表达系统 PDE 以及这些酶的耐药突变体的遗传选择。 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。 我们现在建议审查 利用改进的方法，确定几种 PDE 类型中的同工酶特异性 它建立在我们重组酵母系统的优势之上。 我们将 利用嵌合 PDE 酶来确定哪些残基是足够的， 在偏微分方程的背景下，赋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 优化耐药突变体的选择，导致 识别解释这些特性的特定残基。 还提出了检查底物特异性的实验。 结果 将解决同工酶特异性决定簇是否位于 在不同形式的偏微分方程中保守的模式。 此外， 从这些研究中得出的信息应该对以偏微分方程为中心的有用 同工酶之间的区别至关重要的治疗方法。 它还可以服务 作为分析其他复杂同工酶家族的模型。