ABSTRACT
Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.
IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.
INTRODUCTION
The Filoviridae family consists of Ebola virus (EBOV) and Marburg virus (MARV), which can cause severe hemorrhagic fever in human and nonhuman primates with mortality rates of up to 90%. The viral outbreaks are sporadic and unpredictable and so far have been limited to Africa (1). It is thought that fruit bats are the natural reservoirs of EBOV (2). Although several vaccines and therapeutic options have been developed and shown to be effective in nonhuman primate models (3–5), there is currently no vaccine or treatment approved for filoviral infections in humans. The 2013-2015 West Africa Ebola epidemic, with more than 25,000 people infected and more than 12,000 deaths, underlines the global challenge of treating and controlling the infections and diseases associated with these viruses.
One of the potential targets to block filoviral infection is at the entry step, which is mediated by a single viral glycoprotein (GP). GP is composed of two subunits, GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. GP on the surface of virions is present as a homotrimer of GP1/GP2 heterodimer (1). Following the initial attachment of virions to the host cell surface, likely through the interaction of GP with heparan sulfate and other closely related glycosaminoglycans (GAGs) (6, 7), virions are believed to enter the cell by a process of endocytosis into the endosome, where fusion of virus and cell membranes occurs. Although numerous other host factors have been implicated in Ebola/Marburg virus entry, the entry mechanism of filovirus is still poorly understood.
In this study, we demonstrate that several classes of G protein-coupled receptor (GPCR) antagonists can effectively inhibit entry of both EBOV and MARV. This finding has important implications in our understanding of the role of GPCRs in filoviral entry and in the development of GPCR antagonists as a potential antifiloviral therapeutic option.
MATERIALS AND METHODS
Cell culture and virus.
Human 293T embryonic kidney cells and human A549 lung epithelial cell lines were cultured in Dulbecco's modified Eagle medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 μg/ml of streptomycin, and 100 units of penicillin (Invitrogen). Vero E6 cells were maintained in Eagle's minimum essential medium (EMEM, Gibco) supplemented with 10% FBS (Gibco).
The pseudovirions for high-throughput sequencing (HTS) were created by the following plasmids: hemagglutinin (HA) and neuraminidase (NA), isolated from a highly pathogenic avian influenza virus, A/Goose/Qinghai/59/05 (H5N1) strain, Marburg virus glycoprotein, Ebola virus Zaire envelope glycoprotein, and the HIV-1 proviral vector pNL4-3.Luc.R−E−, which was obtained through the NIH AIDS Research and Reference Reagent program. All three types of pseudovirions, HIV/MARV, HIV/AIV, and HIV/EBOV, were produced by transient cotransfection of human 293T cells using a polyethylenimine (PEI)-based transfection protocol. Plasmids encoding MARV GP (MGP), HA/NA, EBOV GP, and replication-defective HIV vector (pNL4-3.Luc.R−E−) were used for transient cotransfection into 293T producing cells. Five hours after transfection, cells were washed with phosphate-buffered saline (PBS), and 40 ml of fresh medium was added to each plate (150 mm). Forty-eight hours posttransfection, the supernatants were collected and filtered through 0.45-μm-pore-size filters (Nalgene). The pseudovirion stocks were stored at 4°C prior to use.
The infectious filoviruses EBOV, enhanced green fluorescent protein (eGFP) Ebola virus (eGFP-EBOV), Sudan virus (SUDV), Marburg virus (MARV/Angola), and Ravn virus (RAVV) were replicated in Vero E6 cells at 90 to 100% confluence. Cells were inoculated with an approximate multiplicity of infection of 0.1 from historical stocks, and the medium was replaced 72 h after inoculation. Cells were monitored for cytopathic effects, and the supernatant was collected once 95 to 100% of the cells had detached from the surface. The cell supernatant was clarified by centrifugation at 1,200 rpm for 10 min at 4°C, and aliquots were placed at −80°C storage until further use. All infectious virus assays were performed at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) at biosafety level 4 (BSL-4) facilities, with personnel wearing positive-pressure protective suits (ILC Dover) fitted with HEPA filters and umbilicated air. USAMRIID is registered with the Centers for Disease Control and Prevention Select Agent Program for the possession and use of biological select agents and has implemented a biological surety program in accordance with U.S. Army regulation AR 50-1 “Biological Surety.”
Compound library and chemicals.
The Prestwick Chemical Library contains 1,200 FDA-approved drugs. The active compounds were selected for high chemical and pharmacological diversity as well as for bioavailability and safety in humans. Three hundred twenty unique compounds were arrayed in a 384-well plate at 10 mM concentration in dimethyl sulfoxide (DMSO), leaving columns 1, 2, 23, and 24 with DMSO. The positive-control drug for this assay, zidovudine (AZT; Sigma), was solubilized at 10 mM in DMSO. The stock solution was diluted to a final concentration of 5 μM for the screens.
Benztropine mesylate, pizotifen malate, and trimeprazine tartrate were purchased from Santa Cruz Biotech; cyproheptadine hydrochloride sequihydrate and heparin were purchased from Sigma-Aldrich; bafilomycin A1 was purchased from Alexis biochemical; serotonin receptors antagonists were purchased from Tocris Bioscience. The compounds were dissolved in DMSO, and aliquots were stored at −80°C until used.
High-throughput screen.
The Prestwick library was screened at 25 μM in duplicate in a 384-well format with a final DMSO concentration of 0.25% to identify a MARV entry inhibitor. Low-passage A549 cells were seeded at the density of 1,000 cells/well in 384-well plates 24 h before infection. In the presence of compounds, A549 cells were infected by HIV/MARV pseudotyped virus, which contains the luciferase reporter gene. Plates were incubated for 48 h, and infection was then quantified by the luciferase activity of infected A549 cells using the neolite reporter gene assay system (PerkinElmer). Virus alone with DMSO was used as a negative control; virus with 5 μM AZT, an HIV reverse transcriptase inhibitor, was used as a positive control. Data were normalized by plate median value, and the criterion of an average >80% inhibition in duplicate wells was applied for picking hits.
The hit compounds were then cherry-picked into 384-well plates and screened against HIV/MARV, HIV/EBOV, or HIV/AIV to validate the primary result and to identify filovirus-specific hits. The cytotoxicity of hit compounds was also examined by the CellTiter-Glo luminescent cell viability assay (PerkinElmer). The signals in the negative-control wells (DMSO) were used to normalize the data.
The hit compounds were serially diluted for 50% inhibitory concentration (IC50) evaluation. IC50s were determined by fitting the dose-response curves against infection of HIV/MARV or HIV/EBOV with four-parameter logistic regression in GraphPad.
Time-of-addition experiment
A549 cells were incubated with HIV/MARV at 4°C for 1 h to allow virus attachment to the cells. Then, virus was removed and cells were washed with cold PBS two times before fresh medium was added. Temperature was shifted to 37°C to trigger virus entry. At different time points of virus entry, heparin (10 μg/ml), cyproheptadine (25 μM), benztropine (25 μM), bafilomycin A1 (100 nM), or AZT (1 μM) was introduced to assess the impact on virus entry. Triplicate wells were used for each time point. Control-infected cell cultures were treated with drug vehicle (DMSO) only. Virus infection was measured 48 h postinfection as described above.
Microscopy.
Pseudotyped Marburg virus was produced by cotransfection of plasmids encoding MARV GP, replication-defective HIV vector (pNL4-3.Luc.R−E−), and Vpr-GFP. GFP-tagged virions were collected 48 h postinfection, filtered through 0.45-μm-pore-size filters, and then concentrated by ultracentrifugation in a 20% sucrose cushion and suspended in cell culture medium. A549 cells were cultured on glass coverslips and were incubated with GFP-tagged Marburg pseudovirions at 4°C in the presence of benztropine (25 μM), cyproheptadine (25 μM), heparin (10 μg/ml), or DMSO control at 4°C for 15 min. Virus was then removed, and cells were washed twice with cold PBS and cultured for 2 h at 37°C in the continued presence of drugs or control. The cells were washed with cold PBS, fixed, and stained for nuclei using DAPI (4′,6-diamidino-2-phenylindole). All images were taken using a Zeiss laser scanning microscope (LSM) 710 with a 60× objective and analyzed by ImageJ.
Infectious virus assays.
Experiments using live filoviruses were performed in BSL-4 facilities at USAMRIID with personnel wearing positive-pressure protective suits (ILC Dover, Frederica, DE) fitted with HEPA filters and umbilically fed air.
Wild-type-like recombinant EBOV (1976 Mayinga variant)-eGFP and MARV-Angola were used to infect Vero E6 cells. Compounds at a final concentration of 20 μM to 0.08 μM (2-fold serial dilution) were applied together with the viruses, and their antiviral effects were employed to calculate IC50s. Also, the cytotoxicity of these compounds to the Vero E6 cell line was evaluated, and cytotoxicity concentrations (CC50s) were calculated accordingly.
Data analysis.
The screen data were exported as comma-separated-values files and were analyzed using the statistical programming language R (8). An R script was employed to calculate the median of luminescence signals of all samples in each plate, which was used to normalize data. The percentage of inhibition (% inhibition) was calculated as 100 × (1 − normalized signal).
The hit compounds were clustered based on structure similarity using Tanimoto scores calculated from the two-dimensional (2D) structure fingerprint, which was obtained from the online structure clustering service of PubChem. The score matrix was then visualized by an R script.
The mechanism-of-action information of compounds was extracted from the data file from the Prestwick library. The function enrichment analysis of hit compounds was performed based on the information using a Fisher exact test in R.
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