Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based fluorescent polarization (FP) assay for high throughput screening (HTS) of PKD inhibitors. The assay we are proposing is in a 384-well, small volume format and has been adapted for high throughput screening. The research plan has been designed to achieve an important overall objective, specifically the discovery of novel potent and selective small molecule inhibitors of PKD that will be helpful in understanding the biological relevance of PKD and have the potential for long-term therapeutic application. Our preliminary study provided strong data on assay optimization and quality controls. Our proposed primary assay has passed extensive logistical analysis and variability assessment, and has been used successfully to screen a small compound library in HTS mode. Based on this solid foundation of preliminary data, we propose a detailed primary screening assay protocol with a cost estimate and outline the secondary and tertiary screening assays to identify and validate novel selective small molecule inhibitors of PKD. Successful completion of this application will have a profound impact on the advancement of research on diacylglycerol signaling and on the treatment of diseases caused by the deregulation of this signaling network. PKD plays an important role in the pathogenesis of cardiac hypertrophy and cancer in multiple organ systems. Inhibitors of PKD will facilitate the understanding of the relevance of PKD to these pathological conditions and could serve as potential drugs for the treatment of the diseases. ? ? ?
