Phosphorylation Biosensors for Drug Discovery

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DESCRIPTION: Protein kinases are chemically tractable drug targets, yet less than 5% of the human kinome has been thoroughly explored with selective small molecule inhibitors to demonstrate their therapeutic utility. Pharmaceutical companies are interested in evaluation of the therapeutic potential of targeting kinases to define new opportunities to address unmet medical needs. Lucidicor, Inc. has identified and extensively engineered an unusual marine-derived protein, termed PhosFluor", which varies its fluorescence upon phosphorylation. We have extensively engineered native PhosFluor" using Protein Kinase A as a prototype, and are developing this into a cell-based fluorescence assay that provides real-time in vivo visualization of protein phosphorylation. This platform can be applied to virtually any of the 518 protein kinases of the human kinome. The objective of this application is to optimize our novel cell-based platform to respond to protein kinase inhibitors. The working hypothesis is that PhosFluor" can easily be engineered to fluoresce in response to other protein kinases, and can therefore be used monitor the action of compounds that have been developed to modulate protein kinases. Our experimental approach will be to create additional PhosFluor" derivatives by converting engineered phosphorylation sites into substrate sequences for a tyrosine kinase (Fyn kinase) and a cyclin-dependent protein kinase (cdk5). The rationale for the proposed research is that many well-characterized compounds have already been developed for these kinases, and that the successful testing of these biosensors will enable us to determine the effectiveness of our screening method. The proposed research is significant, because it is offers a novel cellular screening platform for new drugs to target kinases and phosphatases, which may result in innovative treatments for disorders such as cancer, inflammation, pain and neuropsychiatric disorders. Finally, this new technology will be convertible to a wide array of protein kinases, greatly expanding the number of drug targets that may potentially be screened.