The overall goal of this research program is to design and develop chemical probes that may be exploited to enhance our understanding of the mechanism of action of bacterial and mammalian isoenzymes of the phospholipase C (PLC) class. Substrate analogues that may serve as potential mechanistic probes and inhibitors will be the synthetic targets of these efforts. Inasmuch as it is known that the mammalian phosphoinositide-specific PLC plays a key role signal transduction by releasing the second messengers 1,2-diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3), these studies should enable elucidation of certain mechanistic features of this important process. Compounds prepared during these studies could have beneficial impact in a variety of disease areas, including anticancer, cardiovascular, and anti-inflammatory. The principal foci of these investigations will be to: (1) develop efficient, general methods for the syntheses of all classes of phospholipids and derivatives thereof that contain modified head groups and/or modified phosphatidic acid subunits; (2) design and prepare rationally selected phospholipids to test hypotheses regarding the mechanism of enzymatic hydrolysis of the phosphodiester bond in different classes of phospholipids; (3) design and synthesize phospholipid substrate analogues for biological screening as potential inhibitors of bacterial and mammalian PLC isoenzymes; and (4) collaborate in single crystal X-ray studies of inhibitors complexed with bacterial PLC to examine phospholipid- enzyme interactions thereby gaining insights to design second generation mechanistic probes. Biological assays and screening experiments to survey structure-activity relationships with bacterial PLC (B. cereus) and PI-PLC (B. thuringiensis) will be executed in our laboratories according to standard protocols. The X-ray crystallographic studies of inhibitors of bacterial PLC will be carried out in collaboration with Professor Edward Hough (University of Tromso, Norway). The in vitro screening for structure-activity relationships with mammalian PI-PLC and PIP2-PLC will be performed in collaboration with scientists at DuPont (Dr. Pat N. Confalone's group), with Professor Philip J. Majerus (Washington University School of Medicine) and Dr. Sue Goo Rhee (National Institutes of Health, Heart, Lung and Blood Institute).
