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PublisherThe University of Arizona.
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AbstractChemical inducers of dimerization (CID) represent an important tool that has been implemented in numerous biological applications namely protein functions, protein stability, signal transduction, gene transcription, etc. Most generally CIDs are defined as bivalent molecules capable of inducing proximity between two targeted proteins. This proximity can in turn promote or disfavor a certain biological activity. Cell permeable small molecules in particular represent a very effective method to induce precise temporal and spatial control over a specific biological target. Our lab has devoted much effort in studying and elucidating the activity and functions of protein kinases, which represent a very attractive therapeutic target for the treatment of cancer and many other disorders. Towards this goal we have developed a general CID enabled three-hybrid split-luciferase methodology for the investigation of kinase-inhibitor interactions in vitro. We demonstrate that by modulating the kinase-ligand affinity of the CID we are able to successfully profile many structurally non-related protein kinases. We also investigate the use of weaker affinity kinase ligands to allow competitive displacement of CID by the selected inhibitor. In addition we report the design, synthesis and applications of novel CID's for the profiling of kinase inhibitors in mammalian cells and we demonstrate the feasibility of the assay to be used as a new platform for the discovery of cell permeable kinase inhibitors. Finally, we report a new ligand-gated split-kinase that can be selectively activated by photocleavable inducers of dimerization. We further prove how the activity of split-proteins can be deactivated with temporal control with use of non DNA damaging UV radiation.
Degree ProgramGraduate College