The phosphoinositide-specific phospholipase C (Pl-PLC) and the phosphatidylinositol 3-kinase (Pl 3-kinase) are key enzymes m the intracellular signal transduction pathway. These enzymes utilize the phosphoinositides, in particular phosphatidylinositol-4,5-bisphosphate [PI (4,5)P2] as substrate. Stimulated hydrolysis of Pl (4,5)P2 by Pl-PLC generates the two second messengers inositol- I ,4 5-trisphosphate (IP3) and sn- 1,2-diacylglycerol (DAG) which are implicated in many physiological responses inciuding mitogenesis. Pl 3-kinase associates with and is phosphorylated by activated growth factor receptors and or ogene products which manifest protein-tyrosine kinase activity. It phosphorylates Pl(4,5)P2 specifically at the D-3 hydroxyl to produce Pl(3,4,5)P3 which is the putative novel and critical second messenger of growth signals. The overall objective is to develop novel biochemical probes of mammalian Pl-PLC and Pl 3-kinase, based on structural analo$ues of the phosphoinositides carrying reporter groups. During Phase 1, the molecular design, synthesis, and characterlzation of a novel set of conjugands capable of linking with reporter groups will be addressed. In addition, prototype photoaffinity probes will be prepared. These probes will be evaluated as substrates and inhibitors in preliminary assays in collaborative studies with other laboratories. During Phase 11, the design of the probes will refined and studies on Pl-PLC and Pl 3-kinase will be extended to identify the substrate binding and catalytic domains, to trace the downstream targets of P 3-kinase products, and to develop substrate analogues and inhibitors suitable for application in structure-based small molecule drug development.Awardee's statement of the potential commercial applications of the research:The phosphoinositide-based novel set of conjugands and the derived fluorescent, photoaffinity, and related reporter probes, as well as the potential affinity chromatography matrices, have important applications in research. In addition, these molecules have potential applications in the newer technique of structure-based rational drug design and discovery for physiological disorders associated with aberrant signal transduction.National Institute of General Medical Sciences (NIGMS)
