| Assay Method Information | |
| | FRET Displacement Assay |
| Description: | The CDK19/CDK8 IC50 values were measured to evaluate activity and to determine CDK19/CDK8 selectivity. The IC50 values of the disclosed compounds was measured using a LanthaScreen europium kinase binding assay (ThermoFisher), as described herein. To a kinase buffer cocktail solution (e.g., 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35) was added: purified recombinant his-tagged CDK19/CycC protein (5 nM), ATP-competitive kinase inhibitor scaffold kinase tracer Alexa Fluor 665 (10 nM), biotin anti-his tag antibody (2 nM), LanthaScreen europium-streptavidin (2 nM). An aliquot of the cocktail solution (10 uL) was added to each well of a LUMITRAC 200: 384 flat bottom, non-treated microtiter white plate. The plate was then covered to protect light sensitive reagents and incubated for 30 min at room temperature to equilibrate before addition of any inhibitors. |
| Affinity data for this assay | |
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