Enzyme Based Reporter Probes of Signal Transduction

The phosphoinositide-specific phospholipase C (PI-PLC) and the phosphatidylinositol 3-kinase (PI3-kinase) are key enzymes in the intracellular signal transduction pathway. These enzymes utilize thephosphoinositides, 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-trisph-osphate (IP3) and sn-1,2-diacylglycerol (DAG) which are implicated in many physiological responsesincluding mitogenesis. PI 3-kinase associates with and is phosphorylated by activated growth factorreceptors and oncogene products which manifest protein-tyrosine kinase activity. It phosphorylatesPI(4,5)P2 specifically at the D-3 hydroxyl to produce PI(3,4,5)P3 which is the putative novel and criticalsecond messenger of growth signals. The overall objective is to develop novel biochemical probes ofmammalian PI-PLC and PI 3-kinase, based on structural analogues of the phosphoinositides carryingreporter groups. During Phase I, the molecular design, synthesis, and characterization of a novel set ofconjugands capable of linking with reporter groups will be addressed. In addition, prototype photoaffinityprobes will be prepared. These probes will be evaluated as substrates and inhibitors in preliminaryassays in collaborative studies with other laboratories. During Phase II, the design of the probes willrefined and studies on PI-PLC and PI 3-kinase will be extended to identify the substrate binding andcatalytic domains, to trace the downstream targets of P 3-kinase products, and to develop substrateanalogues and inhibitors suitable for application in structure-based small molecule drug development.