Time-resolved fluorescence energy transfer (TR-FRET) high-throughput screening assay

TR-FRET technology was used to screen in-house chemical libraries for small-molecule inhibitors of CBP BrD. The final volume of the reaction was 40 μL. The compounds were diluted in CPD buffer with 20 mM HEPES, pH 7.4, and 150 mM NaCl and then transferred to white 384-well plates (PerkinElmer, Waltham, MA, USA, Cat#6007299) and incubated with 10 nM GST-CBP BrD in assay buffer [20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% bovine serum albumin (w/v), 0.01% Triton X-100 (v/v)] at room temperature for 30 min. After incubating with 100 nM H4 substrate peptide [N-C: SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-GGA-K(Ac)-RHRKVGG-K(biotin)] (ChinaPeptides, Suzhou, China) for 30 min, TR-FRET fluorophores, MAb anti-GST-Eu cryptate donor fluorophores and MAb anti-GST-XL665 acceptor fluorophores (Cisbio, Codolet, France, Cat#61GSTKLB and 61GSTXLB), were diluted in assay buffer. Subsequently, 10 μL of the fluorophores was added to each well of the plates and incubated at room temperature for 90 min. Finally, the signals were measured by an EnVision Multilabel plate reader (PerkinElmer; mirror LANCE/DELFIA Dual/Bias, Eu ex 337 nm, Eu em 615 nm; XL665 em 665/10 nm) and analyzed with GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA).