2.3. Biological assay

Human colorectal cancer cell lines including RKO, SW620 and SW480 were purchased from Chinese Academy of Sciences, a typical cell library culture preservation committee (Shanghai, China). Normal human colon epithelial cell line NCM460 was purchased from ATCC (Washington, DC, USA). RPMI-1640 medium (12633020), foetal bovine serum (FBS, 26170043), penicillin-streptomycin (10378016) and phosphate buffered saline (PBS, 70011044) were purchased from Gibco (Eggenstein, Germany). 5-Fluorouracil (5-FU, E0201010050, purity ≥98%) was purchased from Energy Chemical (Shanghai, China). Dimethyl sulfoxide (DMSO, 41639) was obtained from Sigma-Aldrich (St. Louis, MO). 3–(4,5-dimethylthiazol-2-yl)-5–(3-carboxymethoxyphenyl)-2–(4-sulfophenyl)-2H-tetrazolium (MTS, G1111) was purchased from Promega (Madison, WI, USA). All cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere containing 5% CO₂.

Cell viability was measured via MTS assay. RKO, SW620, SW480 and NCM460 cells were seeded in 96-well plates at a density of 5000 cells per well for 24 h. After treatment with the test compounds at indicated concentration for another 48 h or 72 h at 37 °C, MTS solution was added to each well and incubated for another 30 min at 37 °C. Then, the absorbance at 490 nm was measured by a BioTek Synergy H4 microplate Reader (Vermont, USA). The IC₅₀ values were calculated with Graphpad 7.0 software packages.

In brief, compounds were serially diluted in Echo plate according to the plate map and DMSO’s final fraction is 0.1%. Then, transferring compounds and DMSO to 384-well assay plate by Echo. After that, 2× Protein and Peptide Mix was added to the assay plate and 2× Detection Mix was then added to assay plate and shook for 30 s. After incubating the plate at room temperature for 1.5 h, the HTRF signals (Excitation wavelength at 340 nm, Emission wavelength at 615 & 665 nm) was read on EnVision (PerkinElmer, UK). (+)-JQ1 (BPS Bioscience, Cat. No. 27402) was used as a positive control. BRD4-1 (RD-11–157) and BRD4-2 (RD-11–158) were purchased from Reaction Biology Corp. (PENN, USA). For Curve fitting, the inhibition values was calculated in Excel using Equation (1): Inhibition %=(Max-Signal)/(Max-Min)*100, and the IC₅₀ values was calculated in XL-Fit using Equation (2): Y = Bottom + (Top-Bottom)/(1+(IC₅₀/X)*HillSlope), Y is %inhibition and X is compound concentration.

SW620 and SW480 cells were seeded in 6-well plates (1000 cells per well) and cultured in RPMI-1640 medium containing 10% FBS overnight. After the cells treatment with DMSO or compound ID-11 (4, 8, 16, and 32 μM) for 24 h, the culture medium was replaced by fresh RPMI-1640 medium (containing 10% FBS) and continue culturing for another 10 days. Then the cells were washed with PBS and fixed with methanol for 15 min and stained with 0.1% crystal violet for another 30 min. Finally, the cells were washed with running water, air-dried and visualised by photographing.

Cells were seeded in 6-well plates (40,000 cells per well) and cultured in RPMI-1640 medium containing 10% FBS overnight. Then the cells were exposed to DMSO or compound ID-11 (8 or 16 μM) for 24 h. After that, cells were collected, washed with cold PBS for twice and fixed in 75% ethanol overnight at 4 °C. Subsequently, ethanol was removed and suspended in PBS for twice, and then RNase A and propidium iodide (PI) (Multi Sciences, Hangzhou, China) was added and keep in the darkness for 30 min. Finally, the percentages of cells in different cell cycle phases were measured using a FACS Calibur flow cytometer (Bectone Dickinson, San Jose, CA, USA).

Cell apoptosis was determined using Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, New Jersey, USA) in according with manufacturer's instruction. Briefly, cells were cultured in 6-well plates (40 000 cells per well) till reaching logarithmic growth phase and then treatment with DMSO or compound ID-11 (8 or 16 μM) for 24 h. Afterwards, the cells were harvested, washed with PBS for twice and stained with 100 μL of the mixture of Annexin V/FITC and PI in binding buffer (v/v, 5: 5: 100) in the darkness for 30 min. Another 200 μL binding buffer was added before detection. The apoptotic cells were then analysed by FACS Calibur flow cytometry immediately.

Cells were cultured in 6-well plates at a density of 40,000 cells per well overnight, and then exposed to selected compounds at different concentrations for 24 h. Subsequently, the cells were lysed in lysate buffer containing 50 mM Tris-HCl (PH 6.8), 2% SDS, 0.1% bromophenol blue, 1.5% DTT, 10% glycerol. The total protein extracts were boiled, sonicated, separated by 10–12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Then the membranes were blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies at 4 °C overnight. After that, membranes were washed with 1× TBST for three times and incubated with secondary antibody at room temperature for 2 h. Finally, after washing with 1× TBST for three times, the bolts were visualised by an enhanced chemiluminescence kit (Beyotime, China) using the Amersham Imager 600 system (GE Healthcare Life Science, Shanghai, China).

Cells (50,000 cells per well) incubated in 6-well plates were treated with compound ID-11 for 24 h. After that, the total RNA was extracted using Total RNA Extraction Reagent (Invitrogen, USA) according to the manufacturer’s instructions and quantified with NanoDrop 2000 (Nanodrop, USA). Then the cDNA was synthesized from total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). The SYBR Green Master reagents (Applied Biosystems, USA) was used to determine the relative expression of mRNA level by quantitative real-time PCR (qRT-PCR). The primer sequences are shown below.

The cocrystal structure of BRD4 with (+)-JQ1 (PDB: 3MXF) was downloaded from Protein Data Bank (https://www.rcsb.org/) and the whole molecular docking process were carried out in the Ledock software (https://www.lephar.com/). The receptor was prepared using the LePro module with default parameters and a receptor grid was defined as the ligand-binding site based on the cocrystallised ligand and an enclosed box that was in similar in size to the cocrystallised ligand were used to fit the compounds to be docked. The ligand was prepared using ChemBio3D Ultra 14.0 and minimised to the lowest energy. Before the docking study started, the native ligand (+)-JQ1 was re-docked into the binding site using the same set of parameters as described above. The RMSD of the best-docked pose (binding energy=‒8.66 kcal/mol) was 0.628 Å, thus the docking method using Ledock was suitable. Then the docking work between the receptor and ligand was conducted in the LeDock module and the best docking pose was selected as the dominant conformation. The final docking results were processed with PyMOL software (Version 2.1.0).

Molecular properties including absorption, distribution, metabolism and excretion (ADME), toxicity and drug-likeness property of compound ID-11 were predicted at related available website (https://preadmet.bmdrc.kr/).

Data were presented as mean ± standard deviation (SD). One-way ANOVA was used to analyse the statistics by GraphPad Prism 7.0 packages. p < 0.05 was considered as statistically significance.