| Assay Method Information | |
| | In Vitro Enzymatic Activity Assay for Compounds on RETV804M |
| Description: | The compounds in this invention inhibited the enzymatic activity of RETV804M with IC50 values determined by homogeneous time-resolved fluorescence (HTRF) assay. The compounds were diluted 5-fold with 100% DMSO starting from 0.2 mM (7 concentrations in total), and 2 μL of each concentration was added to 48 μL of reaction buffer (50 mM HEPES pH7.5, 0.1 mM Na3VO4, 5 mM MgCl2. 1 mM DTT. 0.001% Tween 20, 20 nM SEB (purchased from Cisbio), and 100 μg/mi BSA) for dilution and mixing. 2.5 μL of compounds and 5 μL of GST-RETV804M (658-1114 aa, final concentration of 0.02 nM) were added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), the mixture was mixed by centrifugation, and 2.5 μL of ATP (final concentration of 1.5 μM) and TK Peptide Substrate mixture (final concentration of 1 μM, purchased from Cisbio) were added to start the reaction, with a total reaction volume of 10 μL. The 384-well plate was placed in an incubator and reacted at 23° C. for 1 h before the reaction was stopped by the addition of 5 μL of TK Antibody (purchased from Cisbio) and 5 μL of Streptavidin-XL665 (purchased from Cisbio). After further incubation in the incubator for 1 h, fluorescence values (excitation at 320 nm, detecting emitted light at 665 run and 620 nm, and a ratio of the two as an enzyme activity signal) were read on Envision (purchased from PerkinElmer). The enzymatic activity signal of RETV804M was measured at 7 concentrations for each compound and the data were calculated using GraphPad Prism software to obtain the IC50 value for the compound. |
| Affinity data for this assay | |
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