| Assay Method Information | |
| | In Vitro Inhibitory Activities on BTK |
| Description: | The half inhibition concentrations (IC50 values) of the compounds disclosed herein on Btk were determined at both enzymatic level and cellular level: the ability of the compounds to inhibit the activity of Btk kinase was determined in an enzymtic activity assay, and the inhibitory effect of the compounds on BCR-induced calcium flux in cells was determined in a cellular function assay.A platform for determining the Btk kinase activity was established using a Homogeneous Time-Resolved Fluorescence (HTRF) methodology, and the activities of the compounds were determined. The compounds were gradiently diluted 3 folds starting from 1 mM with 100% DMSO (totally 11 concentrations). 4 μL of each sample with a different concentration was added into 96 μL of reaction buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 0.005% BAS, 2 mM DTT). 2.5 μL of each solution was added to a 384-well plate (OptiPlate-384, PerkinElmer), followed by adding 5 μL of Btk kinase (Millipore). The mixture was centrifuged to mix well, followed by adding 2.5 μL of a mixture of ATP (final concentration designated as Km) and TK petide (HTRF KinEASE-TK, Cisbio) to initiate the reaction (total reaction volume being 10 μL). The 384-well plate was put in an incubator and the reaction was conducted at 23° C. for 120 min, followed by adding 5 μL of Eu3+ cryptate-labeled anti-phosphotyrosine antibody (Cisbio) and 5 μL of Streptavidin-XL-665 (HTRF KinEASE-TK, Cisbio) to terminate the reaction. After incubating in the incubator for 1 hour, the fluorescent value was read on Envision (PerkinElmer) (excited at 320 nm, and the emitted light was detected at 665 nm and 615 nm, the ratio therebetween being the enzymatic activity). The enzyme activity for each compound was determined at 11 concentrations, and IC50 values were obtained by GraFit Software 6.0 (Erithacus Software).The ability of the compounds to inhibit the release of calcium from intracellular calcium reservoir was determined by calcium flux using Fluo-4 Direct Calcium Assay Kits (Invitrogen) operated on FlexStation III (Molecular Devices) according to manufacturer's instructions. The specific procedures were as follows. Romas cells were cultured in RPMI-1640 (Invitrogen) supplemented with 10% FBS (Hyclone), centrifuged and washed, and re-plated in low serum medium in a 96-well plate (1×105 cells per 45 μL per well), followed by adding 45 μL of fluorescent dye (Invitrogen) and incubating at 37° C. for 1 hour. Compounds to be assayed were gradiently diluted 3 folds with DMSO and then diluted 100 folds with low serum medium. 10 μL of each sample was added to a 96-well plate (Corning) containing cells (final concentration of DMSO was 0.01%). The 96-well plate (Corning) was incubated in an incubator (37° C., 5% CO2) for 30 min. The compound-treated cells were stimulated with a goat anti-human IgM antibody (10 μg/ml; SouthernBiotech) and the fluorescent value was read in FlexStation III (excited at 494 nM and detected at 516 nM for 90 seconds). The data for each compound were fitted using GraphPad Prism 5 (GraphPad Software) and calculated to give corresponding IC50 values. |
| Affinity data for this assay | |
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