Molecular docking study and antiviral evaluation of 2-thioxo-benzo[g]quinazolin-4(3H)-one derivatives
© Al-Salahi et al. 2016
Received: 26 November 2015
Accepted: 7 April 2016
Published: 19 April 2016
The persistent appearance of viral strains that causes a resistant viral infection has led to continuous trials for the design and development of novel antiviral compounds. Benzoquinazoline compounds have been reported to exhibit an interesting antiviral activity. This work aims to study and evaluate the antiviral activity of a newly prepared 2-thioxo-benzo[g]quinazolin-4(3H)-one series against herpes simplex (HSV-1 & 2) and coxsackievirus (CVB4).
The antiviral activity was performed using the MTT assay, in which Vero cells (obtained from the American Type Culture Collection, ATCC) were propagated in fresh Dulbecco’s Modified Eagle’s Medium (DMEM) and challenged with 104 doses of the virus. Thereafter, the cultures were treated simultaneously with two-fold serial dilutions of the tested compound and incubated at 37 °C for 48 h. Molecular docking studies were done on the CVB4 2A proteinase enzyme using Molegro Virtual Docker software.
The cytotoxicity (CC50), effective concentration (EC50) and the selectivity index (SI) values were determined. Based on their EC50 values, a number of the investigated compounds demonstrated weak to moderate activity relative to their parents. Accordingly, compounds 5–9, 11, 15–18, 21, 22, 24, 25, 27 and 28 were active against CVB4, and compounds 5 and 24 were active against HSV-1 and 2 in comparison to ribavirin and acyclovir, which were used as reference drugs.
Herpes simplex (HSV-1 & 2) and Coxsackie B4 (CVB4) viruses belong to the alphaherpesvirinae and picornaviridae families, respectively. In contrast to HSV-1 and 2 which classified as enveloped double-stranded DNA viruses, CVB-4 is non-enveloped RNA viruses. They are common human pathogens and considered a significant worldwide health concern [1–3]. A relatively wide range of diseases, ranging from asymptomatic, mild infections to serious illnesses, are caused by these viruses [4, 5]. In addition, infections by CVB4 have also been known to cause aseptic meningitis, encephalitis, pleurodynia, myocarditis, and pericarditis .
Viral infectious diseases pose a major challenge for modern medicaments because the viruses have high mutation rates, which allow them to escape immune systems and become resistant to the traditional antiviral drugs [6–10]. Furthermore, although the antiviral drugs for diseases caused by several types of viruses such as herpes are available clinically, but the high prevalence of viral infections for which there are no specific treatments or the continuous appearance of new resistant viral strains are serious problems. This make the task of the development of new novel antiviral agents is essential .
Recently, we have reported the biological activity of some prepared triazoloquinazolines against herpes simplex (HSV-1 & 2) and CVB4. However, a number of these prepared compounds were found to possess remarkable and significant antiviral activity [11–13]. Furthermore, synthetic chemistry has shown that benzoquinazoline is a valuable precursor for elaborating many structurally diverse bioactive molecules, particularly as influenza H5N1 and H1N1 antiviral agents [14–17]. In addition, some 2-aminobenzo[de]-isoquinoline-1,3-diones have been reported as antiherpetic agents .
In view of these evidences and an extension of our ongoing research on benzoquinazolines chemistry, we herein report the antiviral evaluation of a new series of 2-thioxo-benzo[g]quinazolin-4(3H)-one derivatives against HSV-1, HSV-2 and CVB4 viruses.
Results and discussion
Synthesized 2-thioxo-benzo[g]quinazolines (7–28)
Antiviral activity against HSVand CVB4 of compounds (1–28) in terms of CC50, EC50 (μg/mL) and SI
Molecular docking results of tested compounds (1–28)
Mammalian cell line
The source and methodology for preparation of the Vero cells were reported in details by Al-Salahi et collaborators . 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 . 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 .
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].
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].
Statistical analysis was done using a one-way ANOVA test . All experiments and data analysis of the antiviral and cytotoxicity evaluations were carried out in RCMB, Al-Azhar University, Cairo, Egypt.
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 . 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) , 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  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 .
RA and MM made a significant contribution to acquisition of data, analysis, manuscript preparation. HAA analysed the data and revised the manuscript. HAG designed and performed the molecular docking study. RE revised and approved the final manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No RG-1435-068.
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