| Assay Method Information | |
| | AlphaLISA |
| Description: | Compounds were diluted by step-down dilution method (final concentration of DMSO was 1%) and added to the wells of a 384 well opti plate at desired concentrations. 5 nM BDR4-BD1 enzyme (produced in-house) and 12 nM of biotinylated substrate were added to the wells, covered and incubated at room temperature (RT) for 1 h. At the end of 1 h 250 ng of GSH acceptor beads were added to the well and incubated for 1 h at RT; then 500 ng of streptavidin donor beads were added and incubated again for 1 h at RT. Plates were read in a Pherastar reader at 680 nm excitation and 570 nm emission. As detailed above, compounds were tested for both BRD4 enzyme inhibitory activities and IC50 were determined. The activities of selected compounds are listed in Table 1 Anticancer activity: Alamar Blue AssayThe impact of the compounds on cancer cell proliferation was determined using the AML cell line MV4-11 (ATCC) in a 3-day proliferation assay. MV4-11 cells were maintained in RPMI supplemented with 10% FBS at 37° C., 5% CO2. For compound testing, MV4-11 cells were plated in a 96-well black bottom plate at a density of 15,000 cells/well in 100 μL culture media and incubated at 37° C. overnight. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 100 μM to 0.005 μM. The DMSO dilution series were then diluted with media, with the final compound concentrations added to the wells ranging from 10 μM to 0.0005 μM. After the additions of compounds, the cells were incubated for 72h and the numbers of viable cells were determined using the Alamar Blue assay (Invitrogen), according the manufacturer suggested protocol. |
| Affinity data for this assay | |
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