MECHANISTIC STUDIES OF PHOSPHOLIPID PHOSPHODIESTERASES

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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).