EFC assay to measure Allosteric Regulation of Protein Kinases

DESCRIPTION (provided by applicant): Kinases mediate the biological actions of neurotransmitters, hormones and growth factors and because of their role in disease, they have been a major target for the development of novel therapeutics to treat cancer, autoimmune disorders, diabetes, and brain diseases. In this grant, we propose to develop a novel technology that will allow for the discovery of highly selective protein kinase drugs targeted to the allosteric regulatory regions of kinases. The technology will measure protein-protein interactions and will employ DiscoveRx's enzyme fragment complementation (EFC) technology, which consists of a sensitive 2-galactosidase (2-gal) complementation approach utilizing two genetically engineered fragments of E. coli 2-gal termed Enzyme Acceptor (EA) and ProLabel. Individually EA and ProLabel show no enzymatic activity, but when co-expressed they can combine to produce an active 2-gal that generates a highly amplified luminescent readout. In order to harness EFC to measure allosteric regulation, we will insert the EA and ProLabel fragments into a protein kinase and its allosteric regulator protein. When the kinase and its partner interact, EA and ProLabel will recombine to form active 2-gal and a luminescent response, which will be lost when a drug blocks the interaction of the kinase with its allosteric regulator. Feasibility assays will focus on developing a cell-based technology to measure interaction of the subunits of cAMP dependent protein kinase (PKA), and the interaction of two members of the MAP kinase family, MEK and ERK2. These feasibility studies will lead to future phase II SBIR studies to develop EFC as a kinome-wide assay system to detect novel allosteric regulators of protein kinases which could provide the pharmaceutical industry with an approach to develop a new generation of protein kinase targeted drugs.