COMPLEMENTATION ASSAY FOR PROTEIN KINASE INHIBITORS

Protein kinases are a family of enzymes that play a pivotal role in signal transduction. Because of their importance in cell function and disease, kinases have become an major target for drug development by the pharmaceutical industry. Based on estimates from the genome project, there are close to 900 protein kinases expressed in humans, the vast majority of which are orphan kinases which can not be detected using commercially available kinase assay. In this grant proposal, we propose to develop a universal protein kinase assay that can be used for high throughput drug screening against any kinase. This assay will be based on a modification of CEDIA technology, which has been used for over 10 years to develop homogeneous assays for the clinical diagnostics market. CEDIA technology is an in vitro complementation reaction, in which inactive, recombinant fragments of E. coli beta-galactosidase recombine rapidly to form active enzyme, which can be detected using chromogenic, fluorogenic or chemiluminescent substrates. The complementation reaction is used as a signaling system for competitive binding assays and has been adapted by us to measure reactions such as ligand binding to receptors, enzymatic reaction such as protease activity and protein-protein inactions. The objective of this phase I SBIR proposal is to develop a simple, sensitive, homogenous CEDIA assay to screen protein kinases for selective inhibitors based on the binding of staurosporine to protein kinases. Staurosporine is a universal, high affinity protein kinase inhibitor which directly interacts with the ATP binding site in kinases. We will develop a competitive binding assay employing this ligand to bind to individual recombinant protein kinases to identify specific inhibitors selected for by their specific interaction with the unique substrate recognition sites of each protein kinase. Initial studies will test the suitability of the CEDIA assay to measure staurosporine binding to protein kinase C. our proof of principle studies will form the basis of future (phase II SBIR) studies to develop a high through put screening assay to identify specific inhibitors against any kinase.