- Split View
-
Views
-
Cite
Cite
Pouya Hassandarvish, Adrian Oo, Amin Jokar, Alexander Zukiwski, Stefan Proniuk, Sazaly Abu Bakar, Keivan Zandi, Exploring the in vitro potential of celecoxib derivative AR-12 as an effective antiviral compound against four dengue virus serotypes, Journal of Antimicrobial Chemotherapy, Volume 72, Issue 9, September 2017, Pages 2438–2442, https://doi.org/10.1093/jac/dkx191
- Share Icon Share
Abstract
With no clinically effective antiviral options available, infections and fatalities associated with dengue virus (DENV) have reached an alarming level worldwide. We have designed this study to evaluate the efficacy of the celecoxib derivative AR-12 against the in vitro replication of all four DENV serotypes.
Each 24-well plate of Vero cells infected with all four DENV serotypes, singly, was subjected to treatments with various doses of AR-12. Following 48 h of incubation, inhibitory efficacies of AR-12 against the different DENV serotypes were evaluated by conducting a virus yield reduction assay whereby DENV RNA copy numbers present in the collected supernatant were quantified using qRT–PCR. The underlying mechanism(s) possibly involved in the compound’s inhibitory activities were then investigated by performing molecular docking on several potential target human and DENV protein domains.
The qRT–PCR data demonstrated that DENV-3 was most potently inhibited by AR-12, followed by DENV-1, DENV-2 and DENV-4. Our molecular docking findings suggested that AR-12 possibly exerted its inhibitory effects by interfering with the chaperone activities of heat shock proteins.
These results serve as vital information for the design of future studies involving in vitro mechanistic studies and animal models, aiming to decipher the potential of AR-12 as a potential therapeutic option for DENV infection.
Introduction
Reported cases of dengue virus (DENV) infections have been on the rise annually as geographical regions affected by this virus, transmitted by Aedes spp., have increased 4-fold over recent decades. DENV is endemic in tropical and sub-tropical countries, and infected patients may be asymptomatic or manifest 7–10 days of symptoms, including high fever, headache and myalgia, whereas secondary infections may lead to the more severe dengue haemorrhagic fever.1 Effective antiviral treatment for the four DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) have remained elusive, with only symptomatic treatments available.
AR-12, also known as OSU-03012, is a derivative of the cyclooxygenase (COX)-2 non-steroidal anti-inflammatory drug celecoxib, which, unlike its parent compound, does not exhibit COX-2 inhibitory activities.2 It has been suggested to act on protein kinases such as PDK1 and p21-activated kinase as well as exerting its inhibitory effects by downregulating the chaperone activities of heat shock proteins (HSPs).3,4 A wide range of preclinical data, such as anticancer, antimicrobial and antifungal activities, have been reported for AR-12.5–7,In vitro antiviral effects of AR-12 against haemorrhagic fever RNA viruses such as Ebola virus, Nipah virus, Lassa virus and Marburg virus were also observed via the reduction in viral RNA yields following treatment with the compound.8 Our study aims to identify the inhibitory effects of AR-12 on the in vitro replication of the four DENV serotypes in addition to predicting the target protein(s) upon which the compound exerts its anti-DENV activities.
Materials and methods
Cells and virus
DENV propagation and subsequent antiviral assays were performed in c6/36 and African green monkey kidney epithelial (Vero) cell lines, respectively. The cells were grown in EMEM (Gibco, NY, USA; catalogue number 61100061) containing 10% FBS (Gibco, NY, USA; Catalogue Number: 10100147). C6/36 and Vero cells were incubated at 28 and 37°C in the presence of 3% and 5% CO2, respectively. Clinical DENV isolates comprising the four different DENV serotypes were extracted from patient samples at the University of Malaya Medical Centre and identified using full-genome sequencing (GenBank accession numbers: DENV-1, FR666924; DENV-2, AJ556807; DENV-3, AB010982; and DENV-4, AJ428557). Virus was titrated following successful propagation and aliquoted for storage at −80°C until needed. The powder form of celecoxib derivative, AR-12, was provided by Arno Therapeutics. Stock solution was prepared by dissolving the compound in DMSO and stored in aliquots at −20°C until needed.
Cytotoxicity assay
Toxicity of AR-12 in Vero cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Following an overnight cell seeding in a 96-well microplate, confluent Vero cells were treated with different concentrations of AR-12 in triplicate and incubated at 37°C for 48 h. Fifteen microlitres of MTS solution (Promega, WI, USA; catalogue number G3581) was added to each well and, after a further 4 h of incubation, the OD of each well was read at 570 nm using a microplate reader (Tecan, Mannedorf, Switzerland).
Evaluation of AR-12’s antiviral activities against all four DENV serotypes
Vero cells were infected with each DENV serotype at moi = 0.1 and incubated at 37°C for 1 h to allow virus attachment. Infected cells were rinsed with sterile PBS twice to remove unbound DENV particles, followed by the addition of different concentrations of AR-12. The plates were left incubated at 37°C in the presence of 5% CO2 for 48 h. DENV RNA was extracted and purified from the supernatant collected for subsequent virus quantification by a one-step qRT–PCR.
Statistical analysis
GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA) was used to generate dose–response curves to evaluate AR-12’s cytotoxicity and antiviral efficacy. Results, presented as mean ± SEM, showing a P value <0.05 were considered to be statistically significant when one-way analysis of variance (ANOVA) tests were performed.
Molecular docking
Protein crystal structures were retrieved from the Protein Data Bank (PDB), namely DENV NS2B/NS3 protease domain (PDB id. 2FOM), DENV NS5 RNA polymerase domain (PDB id. 2J7U), envelope protein (PDB id. 1OKE), as were also human HSP27 core domain (PDB id. 4MJH) and HSP70 ATPase domain (PDB id. 1S3X), whereas the ligand structure was generated using ChemDraw (CambridgeSoft). In preparation for subsequent docking utilizing AutoDock Vina 1.5.6, proteins and ligand were subjected to a few procedures, including the application of CHARMM27 force field using Discovery Studio 2.5. Important data, such as binding affinities and formation of various bonds, were analysed using AutoDock Vina 1.5.6 and Discovery Studio 2.5 from the molecular docking output files, which had been converged by PyMOL.
Results and discussion
For many years, decreasing the amount of DENV-associated morbidity and fatality has been the ultimate aim of researchers, especially for those working on potential therapeutic agents. However, there is yet to be any breakthrough discovery that provides clinical benefit for the millions of DENV-infected patients around the globe. HSPs are host cellular stress proteins that are upregulated in events that occur in stressful environments such as viral infections. HSPs play the role of chaperones, which are involved in the assembly and repair of misfolded proteins and have long been associated with viral infections such as human papillomavirus, herpes simplex virus type 1 and measles virus.9–11 In this study, we examined the potential of the HSP inhibitor AR-12 as an antiviral agent for DENV infections.
A cytotoxicity assay by the MTS method was first performed to determine the viability of Vero cells at different concentrations of AR-12. Intensity of colour change in each well as measured by the plate reader demonstrated 50% cell viability (CC50) at the concentration of 65.9 μM, whereas the maximum non-toxic dose (MNTD) at which 90% of cells were viable was recorded at 16.5 μM (Figure 1a). Our vehicle control, 1% DMSO, did not exhibit any cytotoxicity towards the Vero cell line. The data demonstrated that AR-12 has relatively low toxicity to the cells and hence was deemed suitable for further tests on its antiviral properties. Subsequent antiviral assays were conducted using AR-12 at concentrations lower than the MNTD to ensure good cellular conditions.
The compound was screened for its antiviral activities against all four DENV serotypes and the resulting qRT–PCR readings demonstrated that it is an effective inhibitor of the different serotypes, as viral loads were decreased in a concentration-dependent manner (Figure 1b). Relative to virus control, in which Vero cells were only infected with DENV, highest inhibition was observed for DENV-3 in vitro replication, followed by DENV-1, DENV-2 and DENV-4. Fifty percent of each serotype RNA production was inhibited at AR-12 concentrations of 0.60 μM (DENV-3), 0.64 μM (DENV-1), 0.69 μM (DENV-2) and 0.80 μM (DENV-4), respectively. At 2.5 μM, AR-12 suppressed >70% of RNA production by all four DENV serotypes. Though AR-12 was most potent against DENV-3, differences in efficacies across all serotypes were small. Hence, the compound could be considered as an acceptable candidate drug for evaluation as a potential treatment of symptomatic DENV infections, especially because previous clinical data demonstrated that plasma concentrations of up to 5 μM were achieved. The exact underlying inhibitory mechanism(s) remains unknown as DENV replication involves multiple cellular pathways. However, AR-12 has been recently reported to suppress DENV replication by downregulating the phosphoinositide 3-kinase (P13K)–AKT signalling pathway as well as glucose-regulated protein 78 (GRP78) expression.12 The P13K–AKT pathway promotes DENV replication by counteracting DENV-induced cellular apoptosis, whereas GRP78 was suggested to function as a chaperone as well as a receptor for the virus itself. In this study, we further examined other proteins that may serve as targets of AR-12’s antiviral activity against DENV via computational predictions using molecular docking.
Molecular docking was conducted to analyse the molecular interaction(s) between AR-12 and several protein domains vital for virus replication. As illustrated in Figure 2(a), our ligand binds most strongly to the human HSP27 core domain (4MJH), followed by HSP70 ATPase domain (1S3X), DENV NS5 RNA polymerase domain (2J7U), DENV NS2B/NS3 protease domain (2FOM) and finally DENV envelope protein (1OKE). Ligand interactions with each protein domain were manifested by hydrogen and π bonds formed between specific amino acid residues and the ligand (Figure 2b). The obtained molecular docking results match previous reports of AR-12’s close associations with HSPs.4 Stronger interactions were formed with both HSP domains (4MJH and 1S3X) tested while other viral proteins had relatively lower binding affinities. Loss of HSP27 activities has been linked to autophagosome formation and a reduction in pro-inflammatory mediators, which could disrupt DENV replications and pathogenesis, respectively, whereas the ATPase domain of HSP70 is vital for the protein’s chaperone functions.13,14
On the other hand, DENV NS3 and NS5 proteins contribute to virus replication via their respective roles as helicase and protease as well as RNA-dependent RNA polymerase.15,16 Both proteins also exhibit close interactions with each other as induction of NS3’s triphosphatase activities by NS5 has been reported.17 The structural envelope protein is vital during the early and final stages of the DENV replication cycle. Surrounding pH-mediated conformational changes and glycosylation of ASN67 of the protein are essential for entry and release of infectious virions from host cells.18–20 Inhibition of any of the above proteins, each playing different roles in DENV replication, will certainly reduce virus infectivity.
In conclusion, we have proved that AR-12 exhibits potent in vitro antiviral activity against all four DENV serotypes at concentrations well below the cytotoxic concentrations. Activity against all four DENV serotypes is vital, as from time to time DENV serotypes can vary across geographic regions. The molecular docking studies have demonstrated that AR-12 interacts closely with several proteins required for successful virus replication. Future studies are needed to elucidate further AR-12’s inhibitory pathways and its efficacy in animal models.
Funding
This work was supported by the Ministry of Higher Education Malaysia (MOHE) via its Fundamental Research Grant Scheme (FRGS) (FP054–2014B).
Transparency declarations
None to declare.