MECHANISTIC STUDIES OF PHOSPHOLIPID PHOSPHODIESTERASES

磷脂磷酸二酯酶的机理研究

• 批准号: 2608899
• 负责人: STEPHEN MARTIN
• 金额: $ 15.16万
• 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/2608899
• 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 水解的细菌和哺乳动物同工酶的作用机理 不同类别的磷脂的磷酸二酯键和 发现这些酶的新型抑制剂。主要的重点将介绍 磷脂酶C（PLC）类的细胞内酶。这些 酶参与信号通路，其中细胞反应 产生诸如增生或分泌 细胞外刺激。哺乳动物磷酸肌醇特异性的激活。 通过受体连接G蛋白的PLC导致水解的水解 磷脂酰肌醇-4,5-双磷酸（PIP2）释放第二个 MessengerS L，2-二酰基甘油（DAG）和L，4,5-肌醇三磷酸盐 （IP3）。 DAG激活蛋白激酶C（PKC），IP3从 在内质网中存储。对刺激的持续反应 由任何一个PLC加工磷脂酰胆碱（PC）引起的 直接产生DAG或通过PLD产生磷酸酸（PA）； PA 然后将水解为DAG。在这些过程中合成的化合物 调查可以用作研究生理的工具 干扰这一信号转导步骤的后果， 有些应该是各种疾病地区的潜在候选药物， 包括抗癌，心血管和抗炎。 这些调查的主要重点将是：（l）发展 所有类别的合成的高效，一般方法 磷脂以及那些包含改良头部组的类似物 和/或磷酸酯组的替代； （2）设计和合成 用于生物学筛选的磷脂基板类似物作为抑制剂 细菌和哺乳动物PLC同工酶； （3）合作X射线研究 与天然和突变细菌PLC BC和PI-PLC复合的抑制剂 酶检查磷脂 - 酶相互作用并获得见解 进入不同活性的结合和催化中的特定作用 位点残基并阐明水解机理； （4） 在定向诱变的研究中协作以确认提出的 活性部位残基的催化作用； （5）利用 抑制剂设计和准备的生物活性构象 非基层类似物作为酶抑制剂。 与调查结构活性关系的生物学测定 细菌PLC BC和PI-PLC将使用我们已经有测定 发达。 PLC BC的站点定向诱变正在进行中 与T. Johansen教授（挪威特罗姆索大学）合作，从中 我们已经获得了质粒，以及J. Robertus教授（得克萨斯州大学）。这 PI-PLC的位置定向诱变将与O. H.教授一起进行。 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和单个突变体的复合物 与E. Hough教授（Tromso大学）和Robertus教授合作， 而患有PI-PLC的人将与格里菲斯教授和博士一起执行。 Dirk Heinz（Universitat Freiburg）。