AlphaScreen compound–protein interaction assay

The AlphaScreen assays were performed by Reaction Biology Corp. (Malvern, PA). Recombinant Human N-terminal Histagged BRD4 BD1 and BD2 were expressed in Escherichia coli. The C-terminal-biotinylated histone H4 peptide (residues 1–21) was used as ligand in the assay. In each well of the reaction plate, a mixture of BRD4 BD1 or BD2 and one individual compound in a reaction buffer containing 50 mM HEPES, 100 mM NaCl, 0.05% CHAPS, 0.1% BSA and 1.0% DMSO (pH7.5) was added and pre-incubated for 30 min. C-Terminal-biotinylated histone H4 peptide was added to each well and incubated for 30 min at room temperature with gentle shaking. Then streptavidin-coated donor beads and AlphaScreen Ni acceptor beads (PerkinElmer, Waltham, MA, USA) were added and the plate was sealed and incubated in the dark for 60 min. When excited by 680 nm laser, donor beads generated singlet oxygens, which diffused to acceptor beads and was converted to light signal between 520 and 620 nm that was recorded with an AlphaQuest®-HTS Microplate reader (PerkinElmer, Waltham, MA, USA). IC₅₀ values and curve fits were obtained using GraphPad Prism 7.0 (San Diego, CA, USA).