The phosphoinositide-specific phospholipase C (PI-PLC) and the phosphatidylinositol 3-kinase (PI 3-kinase) are key enzymes in 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 PI (4,5)P2 by PI- PLC generates the two second messengers inositol-1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) which are implicated in many physiological responses including mitogenesis. PI 3-kinase associates with and is phosphorylated by activated growth factor receptors and oncogene products which manifest protein-tyrosine kinase activity. It phosphorylates PI(4,5)P2 specifically at the D-3 hydroxyl to produce PI(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 PI-PLC and PI 3-kinase, based on structural analogues of the phosphoinositides carrying reporter groups. During Phase I, the molecular design, synthesis, and characterization 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 II, the design of the probes will refined and studies on PI-PLC and PI 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.
