Emission Ratiometric Kinase Activity Reports in High Throughput Drug Screening and Analysis

Unmet Need: Genetically-encoded reporters based on fluorescence resonance energy transfer (FRET) have become powerful tools for monitoring the activities of protein kinases and second messengers in live-cell imaging, but their application in high throughput assays has yet to be realized. Protein phosphorylation is one of the major regulatory mechanisms controlling cellular functions and aberrant kinase activities are found to form the molecular basis for many disease phenotypes. As a result, protein kinases are well recognized as important drug targets and are a focus of small molecule drug discovery efforts. There is a need in the art for sensitive PKA reporters which can be used for accurate measurements of spatial and temporal PKA activities in living cells. Advantages of this technology include:

• Reporter molecules to monitor activation and deactivation of PKA with high temporal and spatial resolution
• Emission ratiometric readout suitable for HTP screens in living cells to identify new PKA modulators
• High sensitivity at single cell level and allows targeting to subcellular compartments

 
Technical Details: JHU inventors have developed improved emission ratiometric reporters as research tools for monitoring the activation and deactivation of a prototype kinase, cAMP-dependent kinase (PKA), in living cells after transfection with plasmid constructs encoding the reporter protein, using a Fluorescent Resonance Energy Transfer (FRET) assay. The reporter molecule comprises a pair of fluorescent proteins linked by a region containing a phosphoamino acid binding domain followed by a substrate specific for PKA. Phosphorylation-induced conformational changes result in a change in the FRET emission ratio between the fluorescent protein color variants. Previous FRET-based reporters for PKA suffer from poor dynamic range, making it difficult to visualize subtle changes in PKA dynamics and limiting the applicability of the reporters. The new reporters provide increased sensitivity at the single cell level and can be targeted to subcellular compartments (e.g. plasma membrane and nucleus) while still maintaining improved dynamic range. This approach can be extended to other kinases.

Patent Status: Granted US Patent 8,080,235

Publications/Associated Cases: Biochem Biophys Res Commun. 2006 Sep 22;348(2):716-21., ACS Chem Biol. 2006 Jul 21;1(6):371-6.
 

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