According to the Drug and Market Development Report (2003) 30% of research spending in drug development was focused on kinases. Kinase inhibitors are being sought as targeted therapeutics for diseases from cancer and inflammation to diabetes. Determining efficacy and specificity of the novel compounds in cell-based assays is essential during preclinical investigation. However, the lack of specific physiological substrates for kinases of interest complicates characterization in intact cells. To overcome this complication, Luna Innovations, the University of Virginia and Harvard Medical School propose to develop cell lines stably expressing kinase-specific molecular markers for quantifying catalytic activity of specific kinases of interest in cell-based kinase assays. For this phase I program, we will develop and validate the reagents for two prototype cell-based kinase assays for monitoring activity of the proto-oncogene, Akt2. These prototype assays will be used to monitor the intracellular kinase activity of the exogenously expressed Akt2 and activity of the endogenous Akt. The assays will be developed for use with 96-well streptavidin-coated microplates to immobilize the biotinylated molecular markers. Phospho-specific antibodies will be used to quantify the phosphorylation levels of the immobilized molecular markers. The phase II program will focus on expanding the number kinases for use with the assays and developing the multiplexed format for high content screening. [unreadable] [unreadable] [unreadable]