MECHANISTIC STUDIES OF PHOSPHOLIPID PHOSPHODIESTERASES

磷脂磷酸二酯酶的机理研究

• 批准号: 2022322
• 负责人: STEPHEN MARTIN
• 金额: $ 14.58万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022322
• 关键词: X ray crystallography; active sites; chemical synthesis; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate analog; isozymes; laboratory rabbit; phosphatidylinositols; phospholipase C; phospholipase inhibitor; phospholipids; protein structure function; site directed mutagenesis

The overall goal of this research program is to elucidate the function and mechanism of action of bacterial and mammalian isoenzymes that hydrolyze the phosphodiester bonds of different classes of phospholipids and to discover novel inhibitors of these enzymes. The primary focus will be upon the intracellular enzymes of the phospholipase C (PLC) class. These enzymes are involved in the signaling pathway in which a cellular response such as proliferation or secretion is produced consequent to an extracellular stimulus. Activation of mammalian phosphoinositide-specific. PLC by a receptor-linked G-protein results in the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to release the second messengers l,2-diacylglycerol (DAG) and l,4,5-inositol trisphosphate (IP3). DAG activates protein kinase C (PKC), and IP3 releases calcium from stores in the endoplasmic reticulum. Sustained response to the stimulus arises from processing of phosphatidylcholine (PC) by either PLC, which generates DAG directly, or by PLD, which gives phosphatidic acid (PA); PA is then hydrolyzed to DAG. Compounds synthesized during these investigations may be used as tools to study the physiological consequences of interfering with this step of signal transduction, and some should be potential drug candidates in a variety of disease areas, including anticancer, cardiovascular, and anti-inflammatory. The principal foci of these investigations will be to: (l) develop efficient, general methods for the syntheses of all classes of phospholipids as well as those analogues that contain modified head groups and/or replacements of the phosphodiester group; (2) design and synthesize phospholipid substrate analogues for biological screening as inhibitors of bacterial and mammalian PLC isoenzymes; (3) collaborate in X-ray studies of inhibitors complexed with native and mutant bacterial PLC Bc and PI-PLC enzymes to examine phospholipid-enzyme interactions and to obtain insights into the specific roles in binding and catalysis of the different active site residues and to elucidate the mechanism of hydrolysis; (4) collaborate in studies of site-directed mutagenesis to confirm proposed catalytic roles of active site residues; and (5) exploit the knowledge of the biologically active conformation of inhibitors to design and prepare non-substrate analogues as enzyme inhibitors. Biological assays to survey structure-activity relationships with bacterial PLC Bc and PI-PLC will be executed using assays we have already developed. Site-directed mutagenesis of PLC Bc is being performed in collaboration with Prof. T. Johansen (Univ. of Tromso, Norway), from whom we have obtained plasmids, and Prof. J. Robertus (Univ. of Texas). The site-directed mutagenesis of PI-PLC will be conducted with Prof. O. H. Griffith (Univ. of Oregon). The X-ray crystallographic studies of complexes of inhibitors with PLC Bc and single mutants will be conducted in collaboration with Prof. E. Hough (Univ. of Tromso) and Prof. Robertus, whereas those with PI-PLC will be executed with Prof. Griffith and Dr. Dirk Heinz (Universitat Freiburg).

该研究计划的总体目标是阐明功能和 水解细菌和哺乳动物同工酶的作用机制 不同类别磷脂的磷酸二酯键 发现这些酶的新型抑制剂。主要关注点将是 磷脂酶 C (PLC) 类细胞内酶。这些 酶参与细胞反应的信号传导途径 例如增殖或分泌是由于 细胞外刺激。哺乳动物磷酸肌醇特异性的激活。 PLC 通过受体连接的 G 蛋白导致水解 4,5-二磷酸磷脂酰肌醇 (PIP2) 释放第二个 信使 L,2-二酰基甘油 (DAG) 和 L,4,5-三磷酸肌醇 （IP3）。 DAG 激活蛋白激酶 C (PKC)，IP3 释放钙 储存于内质网中。对刺激的持续反应 由任一 PLC 对磷脂酰胆碱 (PC) 进行处理而产生， 直接生成 DAG，或通过 PLD 生成磷脂酸 (PA)； PA 然后水解成DAG。在这些过程中合成的化合物 调查可用作研究生理学的工具 干扰信号转导步骤的后果，以及 有些应该是多种疾病领域的潜在候选药物， 包括抗癌、心血管和抗炎。 这些调查的主要重点是： (l) 制定 合成所有类别的有效、通用方法 磷脂以及含有修饰头基的类似物 和/或磷酸二酯基团的取代； (2)设计与综合 用于生物筛选的磷脂底物类似物作为抑制剂 细菌和哺乳动物 PLC 同工酶； (3) 合作进行X射线研究 与天然和突变细菌 PLC Bc 和 PI-PLC 复合的抑制剂 酶来检查磷脂-酶相互作用并获得见解 不同活性物质在结合和催化中的具体作用 位点残基并阐明水解机制； (4) 合作进行定点诱变研究以确认提议的 活性位点残基的催化作用； (5) 利用知识 抑制剂的生物活性构象的设计和制备 作为酶抑制剂的非底物类似物。 调查结构-活性关系的生物学测定 细菌 PLC Bc 和 PI-PLC 将使用我们已经掌握的检测方法进行 发达。 PLC Bc 的定点诱变正在进行 与 T. Johansen 教授（挪威特罗姆瑟大学）合作，他的 我们已经获得了质粒，J. Robertus 教授（德克萨斯大学）。这 PI-PLC 的定点诱变将由 O. H. 教授进行 格里菲斯（俄勒冈大学）。 X射线晶体学研究 将进行抑制剂与 PLC Bc 和单一突变体的复合物 与 E. Hough 教授（特罗姆瑟大学）和 Robertus 教授合作， 而使用 PI-PLC 的则将由 Griffith 教授和 Dr. Griffith 执行。 德克·海因茨（弗莱堡大学）。