This application is to develop a HTS assay in response to PAR-10-182. The goal is to identify small molecule probes that disrupt the interaction between the transcription factor RBPJ and SHARP, a scaffold protein that links it to co-repressors and histone deacetylases to repress target genes. Small molecule inhibitors are needed to probe the function of the RBPJ:SHARP interaction in vitro cell culture and in vivo in mice. We present genetic data showing that attenuation of RBPJ [J Recombination signal sequence Binding Protein, aka CSL-1 and Suppressor of Hairless, Su(H)] increases the number of capillaries in the heart, and improves heart muscle function and survival after myocardial infarction (MI). Mechanistically, attenuation RBPJ activates genes that encode angiogenic and cardioprotective factors that might account for the beneficial effects. Although attenuating RBPJ profoundly affects the heart, a number of questions remain unapproachable with current tools, such as timing of action relative to the natural progression of heart disease, dose effects, and whether or not pharmacological inhibition would be an effective therapy to maintain cardiac function. Hence, the small molecule probes will be used in cell culture and animal models of myocardial injury. Prior studies have shown that the SHARP:RBPJ interface is small and likely to be druggable. Our primary HTS assay will identify molecules that disrupt binding between RBPJ and the RBPJ-binding domain of SHARP. For this project, we have already generated a stable cell line for screening that contains a luciferase reporter construct and expresses RBPJ-VP16 and SHARP-GAL4 DNA binding domain fusion proteins. Interaction between these proteins transcriptionally activates the luciferase reporter gene. Preliminary data show a signal to background >40 and the Z'= 0.81.
Aim 1 will develop this into an assay for screening in 384- or 1536 well format with suitable positive and negative plate controls.
Aim 2 will adapt the assay for HTS, develop a secondary TR-FRET assay, as well as counter screens for selectivity.
The Aims i nclude optimizing assay parameters for HTS and development of a critical path for probe development.
There is a large unmet need for novel drug targets for heart disease. Myocardial angiogenesis is considered such a target, but the proteins that coordinate production of the multiple factors needed for normal vessel formation remain unclear. The preliminary data are significant because they indicate that RBPJ is critical for this process;hence, this project is to develop tool compounds to test the physiological effects of pharmacological inhibition of RBPJ.