CK2 and GSK3β kinase inhibition assays

Kinase inhibition assays were performed using Invitrogen's Z′-LYTE™ Kinase Assay Kits – Ser/Thr 11 Peptide (Cat. No. PV3670) and Ser/Thr 9 Peptide (Cat. no. PV3324) for CK2 and GSK3β, respectively. The substrates for the assay of the CK2α recombinant human protein (Cat. no. PV3248) and GSK3β recombinant human protein (Cat. no. PV3365) were procured from Invitrogen. 4,5,6,7-Tetrabromobenzotriazole (TBB) and N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) were used as the standard inhibitor (or positive control) of CK2 and GSK3β, respectively. This assay employs a FRET-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The assay uses a synthetic peptide substrate (the Z′-LYTE™ peptide substrate) that is labeled with a donor fluorophore (coumarin) and an acceptor fluorophore (fluorescein) that make up the FRET pair. The assay uses a ratiometric method to quantify kinase reactions by calculating the ratio of donor emissions to acceptor emissions after excitation of the donor fluorophore at 400 nm. Three assay controls were used along with kinase reactions, the 0% phosphorylation control, 100% phosphorylation control, and 0% inhibition control. The first two controls establish the minimal and maximal emission ratio values, used to calculate the percent phosphorylation achieved in a specific reaction well and 0% inhibition control wells. The first and third controls define the dynamic range in a given assay. Control wells contain no kinase inhibitor. All dilutions were made in kinase buffer A provided with the kits. The final 1× concentration of Kinase Buffer A contains 50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01% Brij-35, and 10 mM MgCl₂. In the kinase reaction, the final 10 μL reaction volume contains 2.5 μL 4× test compound (in 4% DMSO), 5 μL Kinase/peptide mixture (1 : 1) and 2.5 μL 4× ATP solution and the reaction was incubated for 1 hour at 20–25 °C. Then, 5 μL of the development reagent A (a site-specific protease) was added at an optimized concentration, and the assay plate was incubated for another 1 hour at the same temperature. In the next step, 5 μL of the stop reagent was added only for the GSK3β assay. Further, the coumarin (ex. 400 nm, em. 445 nm) and fluorescein (ex. 400 nm, em. 520 nm) emission signals were measured on a fluorescence plate reader (Thermo Scientific Varioskan Flash) for both assays. Percent phosphorylation and percent inhibition were calculated using the following equation: where: emission ratio = ratio of coumarin/fluorescein emission signal intensities from sample wells. C_100% = average coumarin emission signal intensity of the 100% phosphorylation control. C_0% = average coumarin emission signal intensity of the 0% phosphorylation control. F_100% = average fluorescein emission signal intensity of the 100% phosphorylation control. F_0% = average fluorescein emission signal intensity of the 0% phosphorylation control.

We have performed in vitro kinase inhibition assays in two steps. During the primary screening, we have screened all the compounds at a concentration of 11 μM. Compounds that showed more than thirty percent inhibition of both kinases at this concentration were selected further for the secondary screening (IC₅₀ determination).