Fluorescence resonance energy transfer (FRET) titrations

I-BET is titrated against BRD2 (200 nM), BRD3 (100 nM) and BRD4 (50 nM) in 50 mM HEPES pH7.5, 50 mM NaCl, 0.5 mM CHAPS in the presence of tetra-acetylated Histone H4 peptide (200 nM). After equilibrating for 1 hour, the bromodomain protein : peptide interaction is detected using FRET following the addition of 2nM Europium cryptate labelled streptavidin and 10 nM XL-665-labelled anti-6His antibody in assay buffer c

I-BET-762 is a highly potent inhibitor of BET with IC50 values of 32.5–42.5 nM [1].

I-BET-762 is found to have high affinity with BET (Kd of 50.5–61.3 nM). It binds to the acetyl-lysine (AcK)-binding pocket of BET and competes with AcK. The structure of I-BET-762 allows it to bind with BET in ratio of 2:1 results in the high affinity of it. I-BET-762 also has high selectivity, it has no interaction with other bromodomain-containing proteins. I-BET-762 is reported to downregulate the expression of the genes which are induced by LPS, thus causing the decreased expression of LPS-inducible cytokines and chemokines. In vivo assay shows that I-BET-762 has the anti-inflammatory potential. Treatment of I-BET-762 can cure the mice which have started to develop symptoms of inflammatory disease [1].

References:
[1] Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung CW, Chandwani R, Marazzi I, Wilson P, Coste H, White J, Kirilovsky J, Rice CM, Lora JM, Prinjha RK, Lee K, Tarakhovsky A. Suppression of inflammation by a synthetic histone mimic. Nature. 2010 Dec 23;468 (7327):1119-23.