Compound (SMILES): CNS(=O)(=O)c1ccc(Oc2ccc(F)cc2)c(-c2cc(C)c3c(=O)[nH]ccn23)c1
InChIKey: WGSGDACVVLDCET-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Inhibitory Activity Assay of BRD4 Small Molecule Compounds: AlphaScreen kit (Perkin Elmer) was used to detect effect of the compounds on binding of BRD4 bromodomain to acetylated histone H4 polypeptide. The bromodomain BRD4_BD1 (49-170) protein constructed by recombination (Org. Biomol. Chem., 2017, 15, 9352-9361) has a purity greater than 95%, and contains hexahistine tag, namely (His)6tag (hereinafter referred to as (His)6tag), at the N-terminus of the amino acid sequence thereof. Each of fusion proteins containing (His)6tag can be recognized and bound by Ni2+. The acetylated histone H4 polypeptide was provided by Suzhou Qiangyao Biological Technology Co., Ltd, and the sequence was N-C: SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-GGA-K(Ac) RHRKVGG-K (biotin), in which the lysine at positions 5, 8, 12 and 16 was acetylated, and the C-terminus of polypeptide was marked with biotin 5-(2-oxohexahydro-H-thieno[3,4-d]imidazole-4-yl)pentanoic acid, and the purity is greater than 95%. Donor microbeads and acceptor microbeads were purchased from Perkin Elmer (AlphaScreen Histidine Detection Kit (Nickle Chelate) 6760619M Lot: 2236078). The donor microbeads were coated with streptavidin and can bind to biotinylated acetylated H4 polypeptide. The acceptor microbeads were coated with Ni2+ ions and can bind to the BD1 protein with (His)6tag. When the BD1 protein recognized the acetylated H4 polypeptide, the donor microbeads and the acceptor microbeads can be brought closer to a certain distance. Within this range, the donor microbeads can produce singlet oxygen after being irradiated with a 680 nm excitation light, and transfer the singlet oxygen to the acceptor microbeads. After a series of cascade chemical reactions, the acceptor microbeads will generate an emission wave of 520-620 nm, which in turn can be detected a signal. In this experiment, the test system was 20 μL, and the compound needs to be diluted in buffer 1 (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM dithiothreitol). First, a solution of 20 mM compound was consecutively diluted twice, each in 10 times, totally in 100 times to a concentration of 200 μM. Then the solution of 200 μM compound was serially diluted in three times in the buffer 1 containing 1/100 DMSO to obtain a 8× working solution of a compound dilution series with a compound concentration of 200 μM to 10 nM (final concentrations: 25.0 μM, 8.33 μM, 2.77 μM, 0.926 μM, 0.309 μM, 0.103 μM, 0.0343 μM, 0.0114 μM, 0.00381 μM, 0.00127 μM). The positive compound used in the test was JQ (Nature 2010, 468, 1067-1073), which was purchased from Sigma. 2.5 μL of the compound solution was added to a white 384-well plate (OptiPlate-384, PerkinElmer 6007299). The recombinant BD1 protein solution and the acetylated histone H4 polypeptide were diluted in the buffer 2 (20 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Triton X-100, 0.1% bovine serum protein (w/v, Sigma), 1 mM dithiothreitol) to 100 nM and 100 nM respectively, to obtain a 8×BD1 protein working solution and a 4× acetylated histone H4 polypeptide working solution. The donor microbeads and the acceptor microbeads are diluted together in the buffer 2, both in the ratio of 1:100, to obtain a 2× microbead mixed working solution. The plate was added with 2.5 μL of BD1 protein working solution and incubated with the compound for 20 min at room temperature, and then added with 5 μL of acetylated histone H4 polypeptide working solution and incubated at room temperature for 5 min, and finally added with 10 μL of microbead mixed working solution and incubated at room temperature for 60 min. Then the signals were read on an EnVision microplate reader (Perkin Elmer) (excitation wavelength was 680 nM, and detection wavelength was 520-620 nM). IC50 values of the compounds at different concentrations for inhibiting the binding of BD1 protein to acetylated H4 polypeptide were calculated by fitting using GraphPad Prism 5.0 software.
Measurement: IC50 = 82.5 nM (pChEMBL 7.079999923706055)
Descriptors: MW 427.46, ALogP 3.44, HBA 5, HBD 2, PSA 92.67, QED 0.51