Cell-based kinase assay
OwnerSudipta Mahajan, Thomas Hoockinventors
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Abstract
The present invention relates to improved systems and strategies for the investigation of kinase activity in cells. More specifically, cell-based assay methods are provided that allow the phosphorylating activity of a kinase to be determined inside a cell. The invention also provides cell-based screening assays for identifying compounds that have the ability to modulate the phosphorylating activity of protein kinases. Modulators of kinase activity identified by the screening methods are also described, as are pharmaceutical compositions comprising these modulators and methods of using them for inhibiting or enhancing cellular responses triggered by kinase-mediated events.
Claims39
A method for measuring the phosphorylating activity of an enzyme in cells, wherein the enzyme is a kinase catalyzing the phosphorylation of a substrate molecule, the method comprising:
- providing cells in a plurality of wells of a multi-well assay plate;
- exposing the cells to a fluorescently-detectable selective probe such that the probe binds to the phosphorylated substrate;
- measuring the amount of probe bound to the phosphorylated substrate using a Flow Cytometry Plate Reader, wherein the amount of bound probe is proportional to the amount of phosphorylated substrate;
- and
- based on the amount of bound probe measured, determining the phosphorylating activity of the kinase.
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A method for identifying a compound that modulates the phosphorylating activity of an enzyme in cells, wherein the enzyme is a kinase catalyzing the phosphorylation of a substrate molecule, the method comprising:
- providing cells in a plurality of wells of a multi-well assay plate;
- incubating cells in some wells of the assay plate with a candidate compound under conditions and for a time sufficient to allow equilibration, thus obtaining test cells;
- incubating cells in other wells of the assay plate under the same conditions and for the same time absent the candidate compound, thus obtaining control cells;
- exposing the test and control cells to a fluorescently-detectable selective probe such that the selective probe binds to the phosphorylated substrate;
- measuring the amount of selective probe bound to the phosphorylated substrate in the test and control cells using a Flow Cytometry Plate Reader, wherein the amount of selective probe is proportional to the amount of phosphorylated substrate;
- comparing the amount of bound probe in the test and control cells, and
- determining that the candidate compound modulates the phosphorylating activity of the kinase if the amount of bound probe in the test cells is less than or greater than the amount of bound probe in the control cells.
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Dependencies are inferred from the claim text.