Data was collected with biological triplicates of technical triplicates before characterization as a four parameter variable slope fit with GraphPad Prism 8.0. Further, using the concentration of the tracer (0.5 μM) and the Kd of the tracer (0.31 ±0.12 μM), Ki,app was calculated for each compound using the Cheng-Preusoff equation, as shown below. The Kd of the tracer was determined as described previously by kinetically through titrating the tracer against BRD4(D1)-NLuc. NanoBRET NLuc-BRD4(D1) Kinetics. In-cell NanoBRET kinetics assays were performed on HEK293T cells according to transiently expressing a BRD4(D1)-NLuc fusion, according to protocols and materials purchased from the Promega BET Target engagement Kit (Cat. N2131). Briefly, HEK293T cells were seeded into a T25-Corning TC-treated flask at 200,000 cells/well in DMEM containing 10% FBS (Gibco) and 100 μg/mL of penicillin and streptomycin (Gibco). Separately, a transfection cocktail containing 1 μL of NLuc-BRD4(D1) (Promega, 1ug/uL), 9 μL of transfection carrier DNA (Promega, 1 ug/uL), 30 μL of FuGENE (Promega), and 1 mL of OptiMEM media. This transfection cocktail was allowed to equilibrate for 10 min before adding to HEK293T cells, which were then allowed to incubate at 37 °C overnight with 5% CO2. Cells were removed from the plate using 0.05% trypsin (Thermo Fisher Cat.# 25300) and resuspended in OptiMEM media to 200,000 cells/mL and seeded into 15 mL conical tubes with 3 mL aliquots. To these aliquots was added 3 mL of a 1000x compound in DMSO (0.1% DMSO), mixed gently by inversion, and incubated for 2 h at 37 °C with 5% CO2. Tracer solutions were prepared as described in the target engagement assay. After 2 h, cells were centrifuged at 5000 rpm for 5 min and the pellet was resuspended in OptiMEM at 200,000 cells/mL. Cells were then aliquoted into white 96-well plates (Corning) before adding 100 μL of the NanoGlo substrate and extracellular inhibitor cocktail to each well. The donor (410-490 nm) and acceptor (600-610 nm) emissions were collected every 3 min for 180 min, where the mBRET ratio was plotted as a function of time using the equation described in the target engagement assay. Data was collected as biological duplicates of technical duplicates and visualized in GraphPad Prism 8.0.