| Assay Method Information | |
| | In Vitro Enzymatic Activity Assay |
| Description: | In the present application, the IC50 value of the compound of formula (I) in inhibiting BRD4 (BD1) enzyme binding reaction were determined by homogeneous time resolved fluorescence (HTRF). The compound of formula (I) was serially diluted 5-fold with 100% DMSO starting from 0.2 mM (7 concentrations in total), and then 2 μL of the compound of formula (I) at each concentration was added to 48 μL of a reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005 % Tween 20, and 100 μg/mL BSA) for dilution, and mixed well. 2.5 μL of the resulting mixture was added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), then 5 μL of GST-BRD4 (BD1, 44-168 aa) (final concentration: 1 nM) was added, and the resulting mixture was centrifuged, and fully mixed. 2.5 μL of a short peptide Biotin-AHA-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac) RHRKV (final concentration: 100 nM) was then added to initiate the reaction (total reaction volume: 10 μL). The 384-well plate was placed in an incubator at 23° C. to react for 1 hour, and then 5 μL of Eu3+cryptate-labled anti-GST antibody (purchased from Cisbio) and 5 μL of Streptavidin-XL-665 (purchased from Cisbio) were added to terminate the reaction. After being incubated in the incubator for another 1 hour, the fluorescence values (excited at 320 nm, emitted light at 665 nm and 620 nm being detected, and the ratio of the two being the enzyme binding signal) were read on Envision (purchased from PerkinElmer). |
| Affinity data for this assay | |
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