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

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 phenotyes. As a result, protein kinases are well recognized as important drug targets and are a focus of small molecule drug discovery efforts. 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. Description (Set) Proposed Use (Set) These reporters can be used for monitoring the activation and deactivation of a PKA with high temporal and spatial resolution. They can also be used in high throughput assays in living cells for the parallel analysis of a series of pharmacological agonists and antagonists as well as screening of small molecules and other reagents that affect kinase activities. An emission ratiometric readout for kinase activities in high throughput cellular assays have several advantages: 1) cellular assays negate the need to determine all of the appropriate co-factors for the intended targets prior to high throughput screening; 2) cellular assays also ensure that the active compounds already possess adequate solubility, membrane permeability, stability and the ability to act within the biological complexity of the cell; 3) the complexity of cell-based assays may enable discovery of compounds with unique mechanisms of action; and 4) the ratiometric measurement cancels out some sample-to-sample variations and has the potential to provide more consistent readout for screening.

Inventor(s): Zhang, Jin

Type of Offer: Licensing

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