| Assay Method Information | |
| | CDKi Biochemical Assay |
| Description: | Biochemical assays measured the inhibitory effects of compounds in this disclosure on the enzymatic activity of CDK enzyme in complex with Cyclin protein partner by phosphorylation of Ser-780 (S780) on retinoblastoma protein peptide (RB1) in the presence of 1 mM adenosime-5-triphosphate (ATP) and varying concentration of test compound in 20 mM 2-[4-(2-hydroxyethyl)paperazin-1-yl] ethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% bovine serum albumin (BSA), 0.005% Tween 20. Total Reaction volume of 10 μL proceeded for 60 minutes at room temperature (25° C.) and were quenched with 5 μL of 200 mM 2′,2″,2′″-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) pH 8.0 before addition of 5 μL detection solution containing 100 nM fluorophore conjugate streptavidin allophycocyanin (SA-APC), 2 nM Europium labelled Anti-p-RB(S780)-K (Perkin Elmer, 64CUSKAY), 50 mM HEPES, pH 7.5, 400 mM potassium fluoride (KF), 0.1% BSA, and 0.01% Tween-20. Phosphorylation of S780 on RB1 peptide (His-MBP-RB1[773-924]-SEQ ID NO. 9) was detected by TR-FRET after 3 hour incubation with detection solution. Percent phosphorylation activity was plotted against log concentration of compound to generate an apparent ICSO. The following CDK enzyme in complex with different cyclin proteins and protein peptide substrate were used in these assays: CDK1/CyclinB1, Avi-tag, 10 pM used in the assay CDK2/CyclinE1, Avi-tag, 100 pM used in the assay CDK4/cyclinD1, Avi-tag, 20 pM used in the assay CDK6/cyclinD3, Avi-tag, 10 pM used in the assay His-MBP-RB1[773-924], Avi-tag, 200 nM used in the assay |
| Affinity data for this assay | |
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