Computer-aided structure-based virtual screening for CDK2/4/6 triple inhibitors
The chemical structures of a total of 3167 US Food and Drug Administration (FDA) approved drugs were gathered from the ZINC database [25, 26]. The X‑ray crystallographic protein structures used in the studies were obtained from the Protein Data Bank (PDB) [23, 24]. We selected 5 structures of CDK4 and 8 structures of CDK6, and first screened for CDK4/6 inhibitors as described in our previous studies [21, 22]. The co‑crystallized ligands and water molecules were manually removed. The free and open‑source docking docking software idock v2.2.1 developed by our group [27, 28] was used to dock all of the compounds onto all of the ATP binding pocket of all of the CDK4/6 structures using an ensemble docking strategy, to predict their binding conformations and binding affinities as described in previous study . For each compound, idock outputted nine predicted conformations, and the conformation with the best idock score was selected. The 3167 compounds were sorted in the ascending order of their predicted binding free energy averaged across the 13 CDK4/6 structures, and the top-scoring 50 candidate CDK4/6 inhibitors were visually examined using iview. We also collected 14 structures of CDK2 from the 44 CDK2 structures we examined in the previous studies [20,21]. These top 50 candidate inhibitors were then docked onto all of the ATP binding pocket of CDK2 structures. The high-scoring drugs were manually examined based on molecular weight and other drug-like properties. Nine top ranking commercially available compounds were purchased and evaluated.
Monatepil, Fluazuron, Temafloxacin, Ketanserin, Talniflumate, Altanserin, Dutasteride, Mizolastine, Vanoxerine dihydrochloride, 5-Fu were purchased from Sigma‑Aldrich.
Cell lines, cell culture, and experimental conditions
The human HCC cell lines QGY7703 and Huh7 were obtained from Cell bank of Chinese Academy of Sciences (Shanghai, China), and cultured in D-MEM/F-12 medium (GIBCO, USA) containing 8% FBS (Hyclone, Mexico) at 37˚C in 5% CO2 and 95% humidified air. Cells were plated in 96‑, 24‑plates (NEST, China) with medium containing 8% FBS and the test compounds at indicated concentrations (1, 3, 10 and 30μM), and incubated for indicated times (6, 12, 24, 48 or 72 h).
Cell viability MTT and CCK-8 assays
MTT assay were conducted as described in previous studies [20-22].QGY7703 and Huh7 cells were plated at an initial density of 9x103cells/well in 96‑well plates, incubated with MTT (Sigma) reagents and the absorbance measured at 570 nm with a microplate reader (Multiskan Spectrum, Thermo Scientific Microplate Reader, USA). CCK-8 assay was performed as described in the CCK-8 Kit (Dojindo Laboratories). Cells were seeded in 96-well plate, treated with various drugs for indicated time prior to the addition of CCK-8 solution and OD values were measured at 450 nm using a microplate reader.
Cell cycle analysis
The cell cycle profile was determined by Flow cytometry analysis, as described previously [20-22]. Briefly, QGY7703 and Huh7 cells (4x104) were seeded in 24‑well plates in D-MEM/F-12 medium. After 24 h culture, medium were replaced with D-MEM/F-12 containing 8% FBS and vanoxerine dihydrochloride (1, 3, 10 or 30μM) and incubated at 370C for indicated times (6, 12 or 24 h). At the end of experiment, cells were fixed in ice‑cold ethanol, and stained in Coulter DNA‑Prep Reagents (Beyotime Coulter, Beyotime Institute of Biotechnology, Beijing). The cellular DNA content was determined by EPICS xL4 flow cytometer (BD FACSCalibu, USA), and cell cycle distribution determined by BD FACStation software (USA).
QGY7703 and Huh7 cells were seeded in 6-well platein D-MEM/F-12 medium. After 48hours culture, the medium were replaced with D-MEM/F-12 containing 8% FBS and various concentrations of vanoxerine dihydrochloride (1, 3, 10 or 30μM), and incubated at 370C for indicated times (6, 12 or 24 h). Apoptosis was measured by annexinV and propidium iodide (PI) staining (Beyotime Institute of Biotechnology, Beijing) as described in previous studies [20-22].
Western blot analysis
Cells were lysed and Western blotting analysis were performed as described previously [20-22].QHY7703 and Huh7 cells were plated at 6-well plates, cultured in serum starved media (0.125% FBS) at 370C for 24 hours, and then with 10%FBS medium containing various concentrations (3, 10, 30μM) of vanoxerine dihydrochloride. Cells were harvested after 6 hours incubation and proteins analyzed by Western blotting. Primary antibodies were purchased from Cell Signaling Technology, Inc. Danvers, MA, USA). They include anti‑cyclin D1 (no. 2978), anti‑cyclinE (no. 4129), anti‑CDK2/4/6 (no. 2546), anti‑Rb (no. 9313), anti‑phospho‑CDK4, anti‑phospho‑CDK2/4/6 (no. 2561), anti‑Rb (no. 9301), and anti‑GAPDH (no. 5174). As positive controls, three siRNAs targeting each of the CDK2/4/6were designed as described previously , and used to inhibit the expressions of each of the CDK2/4/6proteins in QGY7703 and Huh7 cells. The proteins were measured using enhanced chemiluminescence detection system (Thermo Fisher scientific, USA).
Synergy quantitation of the drug combination study
Synergy quantitation of the drug combination studies were performed according to the Chou--Talalay method. Huh7 cells were plated at an initial density of 5x103cells/well in 96‑well plates, and cells were treated with various concentrations of vanoxerine dihydrochloride and 5-Fu. After 72 hours treatment, cell viability was determined by CCK-8 assay and the absorbance values were measured at 450 nm using microplate reader. The combined effect was analyzed by CompuSyn software (www.combosyn. com), which performs multiple drug dose-effect calculations using the Median Effects methods described by Chou and Talalay to determine the combination index (CI). The drug combinations quantitative definitions of CI are (1) CI=1 represents additive effect, (2) CI < 1represents synergistic effect, and (3) CI > 1 represents antagonism. The formula of the combined index of the two drugs is: CI =(D)1/(Dx)1+(D)2/(Dx)2 , Single dose (D), combined dose (Dx) ) .
Ethic statement and the in vivo nude mice xenografted study
The animal studies were approved by the Kunming Medical University’s laboratory animal ethics committee. Female BALB/C nude mice (4‑5 weeks old, weighing 15 g; Vital River Laboratory Technology Co. Ltd., Beijing, China), were housed and cared under standard conditions (pathogen‑free, 12 h light/dark cycle, 50‑80% humidity, and15‑27˚C) in accordance with guidelines from animal ethics committee in Kunming Medical University. To establish the xenografted model in nude mice (n=20), Huh7 cells (1x106in 0.2 ml PBS) were subcutaneously injected into the right flank, and tumor size measured daily. At seven days after inoculation (tumor volume 80‑100 m3), mice were divided randomly to four groups (5 mice/group) and given daily intraperitoneal injection of (1) vanoxerine dihydrochloride (40mg/kg), (2) 5-Fu (10mg/kg), (3) vanoxerine dihydrochloride (40mg/kg) plus 5-Fu (10mg/kg), (4) control PBS, for 21 days. At the end of experiments, mice were sacrificed by cervical dislocation, tumors excised, weighed, images captured, and immunohistochemistry analysis performed. The tumor volume was calculated by V=ab2/2 (a=longest axis; b=shortest axis).
Tumor tissues were fixed in 10% formalin and embedded in paraffin, sliced into 4 μm sections, deparaffinized, dehydrated, antigen retrieved, blocked with 5% goat serum, and incubated in the primary antibodies: anti-RB1 (1: 500; CST), anti-CDK2 (1:50; Abcam), anti-CDK4 (1: 500; CST), anti-CDK6 (1: 100 Abcam). The slides were washed and incubated with biotinylated anti-mouse or anti-rabbit secondary antibodies. The peroxidase reaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. To quantitate the staining intensity, 5 random fields were chosen, and the numbers of total cells and positive cells were counted in each section under a microscope at 400x magnification. The percentage of positive cell populations from the 5 random fields was analyzed for statistics.
Data were obtained from the triplicates of three different experiments. Values are expressed as the mean ± standard deviation. The dates were analyzed by SPSS software (version 16.0). P<0.05wasconsideredtoindicatestatistically significant difference between values.