All reagents were diluted in the assay buffer (50 mM HEPES-Na + (ChemImpex), 100 mM NaCl (SigmaAldrich), 0.05% CHAPS (RPI), and 0.1% BSA (SigmaAldrich), pH 7.4). The final assay concentrations (after the addition of all assay components) were between 62.5 and 250 nM for the His9tagged bromodomains and the biotinylated peptide were used and optimized depending on the bromodomain. Three-fold serial dilutions were prepared with varying concentrations of the reported compounds and a fixed protein concentration, keeping the final DMSO concentration at 0.1%. To a 384well plate (ProxiPlate-384, PerkinElmer) were added 5 μL of these solutions. The plate was sealed and kept at room temperature for 30 min, followed by the addition of 5 μL of the biotinylated peptide. To each well was added 5 μL of nickel chelate acceptor beads under low light conditions (<100 lx) to a final concentration of 20 μg/mL, and the plate was incubated at room temperature in the dark for 30 min. This was followed by the addition of 5 μL (20 μg/mL final concentration) of streptavidin donor beads in low light conditions. After incubation for 30 min in the dark, the plate was read in the AlphaScreen mode using a PerkinElmer Tecan Spark plate reader. Each compound was run in experimental triplicates of technical replicates, and the data were normalized against the 0 μM inhibitor signal to obtain the percent-normalized AlphaScreen signal. IC50 values were calculated in GraphPad Prism 8 using a sigmoidal four-parameter logistic (4PL) curve fit. Concentrations of peptide and protein for each construct are listen in Supplementary Table .