MECHANISTIC STUDIES OF PHOSPHOLIPID PHOSPHODIESTERASES

这项研究计划的总体目标是阐明功能， 水解细菌和哺乳动物同工酶的作用机制 不同种类磷脂的磷酸二酯键， 发现这些酶的新型抑制剂。重点将放在 磷脂酶C（PLC）类的细胞内酶。这些 酶参与了细胞反应的信号通路 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激活。 PLC通过受体连接的G蛋白导致 磷脂酰肌醇-4，5-二磷酸（PIP2）释放第二个 信使1，2-二酰基甘油（DAG）和1，4，5-三磷酸肌醇 （IP3）。DAG激活蛋白激酶C（PKC），而IP3从细胞中释放钙。 储存在内质网中。对刺激的持续反应 产生于磷脂酰胆碱（PC）的加工， 直接或经PLD生成DAG，生成磷脂酸（PA）; 然后水解为DAG。在这些过程中合成的化合物 研究可以用作研究生理学的工具。 干扰这一信号转导步骤的后果，以及 一些药物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将是： 有效的，一般的方法，用于合成的所有类别的 磷脂以及含有修饰的头部基团的那些类似物 和/或取代磷酸二酯基团;（2）设计和合成 用于生物筛选的磷脂底物类似物， 细菌和哺乳动物PLC同工酶;（3）在X射线研究中合作 与天然和突变细菌PLC Bc和PI-PLC复合的抑制剂 酶来检测磷脂-酶相互作用， 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理;（4） 合作进行定点突变研究，以确认拟议的 活性位点残基的催化作用;（5）利用 抑制剂的生物活性构象来设计和制备 非底物类似物作为酶抑制剂。 用于调查结构-活性关系的生物测定 细菌PLC Bc和PI-PLC将使用我们已经完成的测定来执行。 开发PLC Bc的定点突变正在进行， 与T教授合作约翰森（挪威特罗姆瑟大学），来自 我们已经获得了质粒，J.Robertus教授（德克萨斯大学）。的 将与O. H. 格里菲斯（俄勒冈州大学）。的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教授合作。霍夫（特罗姆瑟大学）和罗伯特教授， 而那些与PI-PLC将执行与格里菲斯教授和博士。 Dirk海因茨（弗赖堡大学）。

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).