NI-57 is an inhibitor of proteins of bromodomain and plant homeodomain finger-containing (BRPF) family, with IC50s of 3.1, 46 and 140 nM for BRPF1, BRPF2 (BRD1) and BRPF3, respectively.

BRPF3:140 nM, BRPF1:3.1 nM, BRPF2 BRD1:46 nM

NI-57通过等温滴定量热法测定，与BRPF1的BRD结合力(Kd)为31 nM，与BRD1的Kd为110 nM，与BRPF3的Kd为410 nM；而与BRD9的结合力较弱(Kd 1000 nM)。NI-57对BRD9(IC50, 520 nM)和BRD4(BD1)(IC50, 3700 nM)、TRIM24(IC50, 1600 nM)的活性影响较小。NI-57还能抑制细胞核内的BRPF BRDs，但对多种癌症细胞系的增殖影响较小，其GI50s分别为10.4 μM（NCI-H1703细胞）、14.7 μM（DMS114）、15.6 μM（HRA-19）和16.6 μM（RERF-LC-Sq1）。此外，NI-57(10 μM)抑制BRPF1可以降低CCL-22基因表达27.7%。

All reagents are diluted in the recommended buffer (50 mM HEPES, 100 mM NaCl, 0.1% BSA; pH = 7.4) supplemented with 0.05% CHAPS and allowed to equilibrate to room temperature prior to addition to plates. 4 mL of HIS-tagged protein is added to low-volume 384-well plates, followed by 4 mL of either buffer, non-biotinylated peptide, solvent or compounds (NI-57, etc.). Plates are sealed and incubated at room temperature for 30 minutes, before the addition of 4 mL biotinylated peptide, resealing and incubation for a further 30 minutes. 4 mL of streptavidin-coated donor beads (25 μg/mL) and 4 μL of nickel chelate acceptor beads (25 μg/mL) are then added under low light conditions. Plates are foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader using an AlphaScreenTM 680 excitation/570 emission filter set. IC50s are calculated in GraphPad Prism 5. Results for compounds (NI-57, etc.) dissolved in DMSO are normalized against corresponding DMSO controls prior to IC50 determination, which is given as the final concentration of the compound in the 20 μL reaction volume.