MECHANISTIC STUDIES OF PHOSPHOLIPID PHOSPHODIESTERASES

这项研究计划的总体目标是阐明其功能和 细菌和哺乳动物同工酶的作用机制 不同种类磷脂和TO的磷二酯键 发现这些酶的新抑制剂。主要关注点将是 磷脂酶C(PLC)类的胞内酶。这些 酶参与了细胞反应的信号通路 如增殖或分泌物是由于 细胞外刺激。哺乳动物磷脂酰肌醇的特异性激活。 PLC由受体连接的G蛋白引起的水解酶 磷脂酰肌醇-4，5-二磷酸(PIP2)释放第二 信使L，2-二酰甘油和L，4，5-三磷酸肌醇 (IP3)。DAG激活蛋白激酶C(PKC)，IP3从 储存在内质网中。对刺激措施的持续反应 产生于由PLC处理磷脂酰胆碱(PC)，该PLC 直接产生DAG，或通过PLD产生磷脂酸(PA) 然后被水解成DAG。在这些过程中合成的化合物 调查可以作为研究生理学的工具。 干扰这一信号转导步骤的后果，以及 其中一些应该是各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：(L)发展 有效的、通用的方法来合成所有类别的 磷脂以及那些含有修饰头基的类似物 和/或取代磷酸二酯基；(2)设计和合成 磷脂底物类似物在生物筛选中的应用 细菌和哺乳动物PLC同工酶；(3)合作进行X射线研究 天然和突变细菌PLC、BC和PI-PLC复合抑制剂的研究 检查磷脂-酶相互作用并获得洞察力的酶 分成不同活性的结合和催化的具体作用 位点残基，并阐明其水解机理； 合作研究定点突变，以确认拟议的 活性中心残基的催化作用；以及(5)利用 缓蚀剂的生物活性构象设计与制备 作为酶抑制剂的非底物类似物。 用于研究结构-活性关系的生物测定法 细菌PLC BC和PI-PLC将使用我们已有的检测方法进行执行 发展起来的。正在进行PLC BC的定点突变 与T.Johansen教授合作(大学挪威特罗姆索)，来自谁 我们已经获得了质粒，J.Robertus教授(大学)。德克萨斯州)。这个 PI-PLC的定点突变将与O.H.教授一起进行。 格里菲斯(大学俄勒冈州)。三元化合物的X射线结晶学研究 将进行缓蚀剂与PLC BC和单一突变体的复合 与E.Hough教授(大学)合作特罗姆索)和罗伯特斯教授， 而那些拥有PI-PLC的人将与格里菲斯教授和Dr。 德克·海因茨(弗莱堡大学)。

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).