Mammalian cell line
The source and methodology for preparation of the Vero cells were reported in details by Al-Salahi et collaborators [11]. The GHSV-UL46, G and E2 viral strains were used for the assay of HSV-1, HSV-2 and CVB4 viruses, respectively.
Evaluation of the antiviral activity
Screening of the antiviral was performed using MTT assay. According to the literature [11, 22, 23], the Vero cells were cultured, then treated with two-fold serial dilutions of the tested compounds, starting from 1000 μg/mL and diluting to about 2 μg/mL (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 μg/mL). Six wells were used for each concentration of the tested compound and three independent experiments were assessed, each containing four replicates per treatment [24]. Untreated Vero cell control and infection controls were made in the absence of tested compounds. Acyclovir and ribavirin were used as positive controls in this assay [25].
After incubating for 48 h, the numbers of viable cells were determined by the MTT test. Briefly, the medium was removed from the 96-well plate and replaced with 100 μL of fresh RPMI 1640 medium without phenol red, then 10 μL of the 12 mM MTT stock solution [5 mg of MTT in 1 mL of phosphate-buffered saline (PBS)] to each well, including the untreated controls. The 96-well plates were then incubated at 37 °C and 5 % CO2 for 4 h. An 85 μL aliquot of the medium was removed from the wells, and 50 μL of dimethyl sulfoxide (DMSO) were added to each well, mixed thoroughly with the pipette, and incubated at 37 °C for 10 min. Then, the optical density was measured at 590 nm with a microplate reader (Sunrise, Tecan U.S. Inc., USA) to determine the number of viable cells [11, 22, 26].
The viral inhibition rate was calculated as follows:
$${\text{Viral Inhibition Rate }} = \left[ {{{\left( {{\text{ODtv }} - {\text{ ODcv}}} \right)} / {\left( {{\text{ODcd}} - {\text{ ODcv}}} \right)}}} \right] \, \times \, 100\;\% ,$$
where ODtv, ODcv and ODcd indicate the absorbance of the tested compounds with virus-infected cells, the absorbance of the virus control and the absorbance of the cell control, respectively. The EC50 was estimated with respect to the virus control from the graphic plots, using STATA modelling software and (SI) calculated from the ratio of CC50 to EC50 [11, 26].
Cytotoxicity evaluation using viability assay
The procedure for seeding and incubation of Vero cells was explained in details in previous research [11, 23, 27]. After the end of the incubation period, the number of viable cells was determined by the MTT test. Briefly, the medium was removed from the 96-well plate and replaced with 100 μL of fresh RPMI 1640 medium without phenol red, then 10 µL of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) to each well including the untreated controls. The 96-well plates were then incubated at 37 °C and 5 % CO2 for 4 h. An 85 μL aliquot of the medium was removed from the wells, and 50 μL of DMSO were added to each well, mixed thoroughly with the pipette, and incubated at 37 °C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader (Sunrise, Tecan U.S. Inc., USA) to determine the number of viable cells. Without added stain, all obtained findings were corrected for background absorbance detected in wells. In the absence of the tested compounds, treated samples were compared with the cell controls. All experiments were carried out in triplicate. The cytotoxicity of each tested compound was calculated [24, 25, 27, 28].
The percentage cell viability, calculated using Microsoft Excel®, is as follows:
$$\% {\text{ Cell Viability }} = \left[ {{{\left( {{\text{Mean Abs}}_{\text{control}} - {\text{Mean Abs}}_{\text{test metabolite}} } \right)} \mathord{\left/ {\vphantom {{\left( {{\text{Mean Abs}}_{\text{control}} - {\text{Mean Abs}}_{\text{test metabolite}} } \right)} {{\text{Mean Abs}}_{\text{control}} }}} \right. \kern-0pt} {{\text{Mean Abs}}_{\text{control}} }}} \right] \, \times { 1}00\;\% ,$$
where Abs equals the absorbance at 590 nm. The STATA statistical analysis package was used for the dose response curve, which was used to calculate CC50.
Data analysis
Statistical analysis was done using a one-way ANOVA test [29]. All experiments and data analysis of the antiviral and cytotoxicity evaluations were carried out in RCMB, Al-Azhar University, Cairo, Egypt.
Molecular docking
The modelling studies were done by a PC with Intel© Core™ i7-3630 QM CPU (2.40 GHz, RAM 8 GB) operating under the Windows 7 Professional Operating System [11]. The modelling processes included several steps: first, download the 3D crystal structures of the Coxsackievirus B4 2A proteinase enzyme with PDB code 1Z8R (Brookhaven Protein Data) [20], and then load this into the Molegro Virtual Docker (MVD 2013.6.0 [Win32]) program (fully functional, free trial version with time limiting license; Molegro Virtual Docker (MVD 2013.6.0), Molegro Bioinformatics Solutions, Denmark, 2013; Thomsen and Christensen, 2006). ChemBio3D Ultra 10 [30] was used to draw the 3D structures of different ligands. Ligands were further optimized using a free version of Marvinsketch 4.1.13 (Marvinsketch, version 6.1.0, Chemaxon, Budapest, Hungary; http://www.chemaxon.com, 2013) with MM force field, and saved in Tripos mol2 file format. MolDock score functions were used with a 0.3 A° grid resolution. Prior to the calculation of the MolDock scores of the tested compounds, the MVD software was benchmarked docking ribavirin [11].