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Journal of Virology logoLink to Journal of Virology
. 2016 Jan 28;90(4):1918–1930. doi: 10.1128/JVI.02030-15

Pan-genotypic Hepatitis C Virus Inhibition by Natural Products Derived from the Wild Egyptian Artichoke

Mahmoud Fahmi Elsebai a,b,c,d, George Koutsoudakis e,*, Verónica Saludes e,*, Gemma Pérez-Vilaró e, Ari Turpeinen b, Sampo Mattila b, Anna Maria Pirttilä d, Fabien Fontaine-Vive c, Mohamed Mehiri c, Andreas Meyerhans f,g, Juana Diez e,
Editor: J-H J Ou
PMCID: PMC4734011  PMID: 26656684

ABSTRACT

Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases. Water extracts of the leaves of the wild Egyptian artichoke (WEA) [Cynara cardunculus L. var. sylvestris (Lam.) Fiori] have been used for centuries in the Sinai Peninsula to treat hepatitis symptoms. Here we isolated and characterized six compounds from the water extracts of WEA and evaluated their HCV inhibition capacities in vitro. Importantly, two of these compounds, grosheimol and cynaropicrin, inhibited HCV with half-maximal effective concentrations (EC50s) in the low micromolar range. They inhibited HCV entry into target cells and were active against both cell-free infection as well as cell-cell transmission. Furthermore, the antiviral activity of both compounds was pan-genotypic as HCV genotypes 1a, 1b, 2b, 3a, 4a, 5a, 6a, and 7a were inhibited. Thus, grosheimol and cynaropicrin are promising candidates for the development of new pan-genotypic entry inhibitors of HCV infection.

IMPORTANCE Because there is no preventive HCV vaccine available today, the discovery of novel anti-HCV cell entry inhibitors could help develop preventive measures against infection. The present study describes two compounds isolated from the wild Egyptian artichoke (WEA) with respect to their structural elucidation, absolute configuration, and quantitative determination. Importantly, both compounds inhibited HCV infection in vitro. The first compound was an unknown molecule, and it was designated “grosheimol,” while the second compound is the known molecule cynaropicrin. Both compounds belong to the group of sesquiterpene lactones. The mode of action of these compounds occurred during the early steps of the HCV life cycle, including cell-free and cell-cell infection inhibition. These natural compounds present promising candidates for further development into anti-HCV therapeutics.

INTRODUCTION

Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus classified as a separate genus (Hepacivirus) within the Flaviviridae family. It shows a high degree of genetic diversity, with 7 major circulating genotypes (1). HCV is mainly transmitted through exposure to HCV-contaminated blood. Most infections remain persistent, summing up to an estimated 150 million chronic HCV carriers worldwide (2). As persistent HCV infection frequently causes chronic hepatitis that can progress to liver cirrhosis and liver cell carcinoma, it is a major threat to human health (3, 4).

Treatment options for chronically infected individuals have dramatically improved over the last few years. This has been due to the development of highly potent direct-acting antivirals (DAAs) that increased sustained response rates to over 90%, even in interferon-free combinations (5). Currently approved DAAs include NS3/4A protease inhibitors (telaprevir, boceprevir, and simeprevir), NS5A inhibitors (daclatasvir and ledipasvir), and the NS5B polymerase inhibitor sofosbuvir. Further antiviral drugs are in clinical trials and are about to be approved. Nonetheless, the rapid replication of HCV, along with the error-prone NS5B polymerase, which lacks proofreading activity, give rise to the generation of mutations throughout the viral genome. This results in the remarkable sequence variation of an infecting HCV population, also known as a quasispecies. Among the members of an HCV quasispecies, drug-resistant mutants are likely to be present and to be able to spread between individuals (6, 7). Furthermore, the current treatments are extremely expensive and thus present a significant burden for health care systems (8). Therefore, there is a continued interest in developing further antiviral drugs with low production costs. As there is no preventive vaccine against HCV infection, another major area of interest is the development of effective preventive measures against infection.

Artichokes are part of the Mediterranean diet and are rich in pharmaceutically active compounds like caffeoylquinic acid derivatives, sesquiterpene lactones, and flavonoids (9). They have been used since ancient times in folk medicine, especially against liver complaints. For example, Bedouins in the Sinai Peninsula (the Asian part of Egypt) use water extracts of the leaves of the wild Egyptian artichoke (WEA) [Cynara cardunculus L. var. sylvestris (Lam.) Fiori] to treat diverse symptoms of hepatitis, like jaundice and ascites. Based on this traditional use, we aimed to explore whether WEA leaf extracts may contain compounds with anti-HCV activities. Here we describe the isolation of 6 natural products from water extracts of WEA. Two of the 6 compounds, cynaropicrin and grosheimol, showed potent antiviral activities against all HCV genotypes and therefore were characterized extensively with respect to their mode of action. Our studies reveal that both compounds interfere with HCV infection of target cells at an early step. Thus, grosheimol and cynaropicrin represent interesting hit compounds for further development of cost-effective anti-HCV drugs.

MATERIALS AND METHODS

Cell culture.

Huh7/Scr cells, Huh7.5.1 Cl.2 cells (kindly provided by F. Chisari, The Scripps Research Institute, La Jolla, CA), and 293T cells (HEK293T cells, CRL-1573; American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml penicillin, and 100 U/ml streptomycin (DMEM complete). Huh7.5/EGFP-NLS-IPS stable cell lines were generated by plasmid transfection with a commercial pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) expressing the enhanced green fluorescent protein (EGFP) followed by nuclear localization signal (NLS [PKKKRKVG]), and beta interferon (IFN-β) promoter stimulator protein 1 (IPS-1) and subsequent selection with G418 as previously described (10). Cells were grown in an incubator with 5% CO2 at 37°C.

Plasmids.

Plasmid pFK-Jc1 has been previously described (11). The subgenomic replicon plasmid pSGR-JFH1 carries a bicistronic construct in which a firefly luciferase gene is expressed via the HCV internal ribosome entry site (IRES) and an encephalomyocarditis virus (EMCV) IRES drives expression of JFH1 nonstructural proteins (NS3 to NS5B) (12). pTN7-Stopp is an HIV plasmid that carries the renilla luciferase reporter gene instead of the nef gene and lacks a functional env gene and thus is set to produce only a single round of infection (13). Plasmid pcDNA3.1-ΔcE1E2-J6CH, which encodes E1E2 glycoproteins of strain HC-J6CH, has been described elsewhere (14). Plasmid pVPack-VSV-G, which encodes the vesicular stomatitis virus glycoprotein (VSV-G), was purchased from Agilent Technologies (Santa Clara, CA). The HCV genotype 1 to 7 plasmids are JFH-1-based reporter virus constructs carrying renilla luciferase inserted at the NS5A gene and structural proteins from all major HCV genotypes (15).

Control compounds for HCV inhibition experiments.

The 2′-modified nucleoside analog 2′-C-methyladenosine (2′-C-Met) was kindly provided by Pablo Gastaminza (Spanish National Biotechnology Center, Madrid, Spain). Dasatinib, erlotinib, and U0126 were purchased from Selleck Chemicals (Houston, TX). For compound stock preparation, each compound was diluted in dimethyl sulfoxide (DMSO) at a final concentration of 10 mM. Chlorpromazine and heparin sodium salt were purchased from Sigma-Aldrich (St. Louis, MO), and stock solutions were prepared in H2O at final concentrations of 50 mg/ml and 100 mg/ml, respectively.

In vitro transcription and electroporation of HCV RNA.

The plasmid carrying the Jc1 construct was linearized with the MluI enzyme, while pSGR-JFH1 was linearized with the XbaI enzyme. Linearized plasmid DNA was purified with the QIAquick PCR purification kit (Qiagen, Düsseldorf, Germany). Purified DNA was subjected to an in vitro transcription reaction with the MEGAscript T7 kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. RNA from the in vitro transcription reaction was purified with the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany), RNA integrity was verified by formaldehyde agarose gel electrophoresis, and the concentration was determined by measurement of the optical density at 260 nm. For RNA electroporations, single-cell suspensions of Huh7.5.1 Cl.2 cells were prepared by trypsinization of cell monolayers. Cells were washed with phosphate-buffered saline (PBS), counted, and resuspended at 1.5 × 107 cells per ml in Cytomix (16) containing 2 mM ATP and 5 mM glutathione. Ten micrograms of in vitro-transcribed RNA was mixed with 400 μl of the cell suspension. Cells were then electroporated, immediately transferred to 10 ml of culture medium, and then a 10-cm dish was seeded with them. The electroporation conditions were 975 μF and 270 V, and electroporation was performed by using a Gene Pulser Xcell system (Bio-Rad, Munich, Germany) and a cuvette with a gap width of 0.4 cm (Bio-Rad).

Generation of HCVcc stocks and determination of virus titers in cell culture supernatants.

For the generation of cell-culture-derived HCV (HCVcc) stocks, supernatants of the electroporated cells were harvested 72 h postelectroporation, cleared by being passed through 45-μm-pore-size filters, and stored at −80°C. For the determination of viral titers, Huh7/Scr cells were seeded at a concentration of 1.2 × 104 cells per well in a 96-well plate in a total volume of 200 μl. Twenty-four hours later, serial dilutions of virus-containing supernatant were added (6 wells per dilution.) Three days later, cells were washed with PBS and fixed for 20 min with ice-cold methanol at −20°C. After three washes with PBS, NS5A was detected with a 1:2,000 dilution of the anti-NS5A antibody 9E10 (kindly provided by C. Rice, The Rockefeller University, NY) in PBS supplemented with 5% bovine serum albumin (BSA) for 1 h at room temperature. Cells were washed again three times with PBS, and bound primary antibodies were detected by incubation in PBS–5% BSA with goat anti-mouse IgG-peroxidase-conjugated antibody (Sigma-Aldrich, St. Louis, MO) at a 1:400 dilution. After 1 h of incubation at room temperature, cells were washed three times with PBS; the Vector NovaRED substrate kit (Linaris Biologische Produkte GmbH, Wertheim, Germany) was used for detection of peroxidase. Virus titers (50% tissue culture infective dose [TCID50] per milliliter) were calculated based on the method described by Spearman and Kärber (54).

Luciferase assays and cell cytotoxicity (viability) assays.

For standard infection assays, 96-well plates were seeded with Huh7/Scr cells at a density of 1.2 × 104 cells/well. One day later, cells were preincubated for 1 h at 37°C with the pertinent compounds and then inoculated with the virus and the compounds for 4 h at 37°C. HCV pseudoparticles (HCVpp) were left for 6 h. Finally, virus-containing medium was replaced by a fresh medium-compound mixture. Firefly and renilla luciferase activities were assayed 72 h postinfection with the Dual-Glo luciferase assay system, while cytotoxicity (viability) assays were carried out with the CytoTox-Glo cytotoxicity assay (both purchased from Promega Corporation, Madison, WI), according to the manufacturer's instructions, using a FLUOstar Optima plate luminometer (BMG Labtech, Ortenberg, Germany). Mean relative light units (RLU) were plotted as a percentage relative to control infections (solvent without compounds) for both infectivity and cell viability. Infections were carried out in duplicates and measured in duplicates as the mean ± standard error of the mean (SEM [n = 4]). The half-maximal effective concentration (EC50) and half-maximal cytotoxic concentration (CC50) were estimated by nonlinear regression of the log inhibitor versus normalized response and used to calculate the selectivity index (SI) value.

Preparation of HCVpp.

HIV-based pseudoparticles bearing HCV glycoproteins were generated by calcium phosphate cotransfection of 293T cells. Briefly, 10-cm dishes were seeded with 3.6 × 106 293T cells 1 day before transfection with equal amounts of pTN7-Stopp and pcDNA3.1-ΔcE1E2-J6CH or pVPack-VSV-G plasmids. A total amount of 20 μg of DNA was mixed with a 2 M CaCl2 solution, and then 2× HEPES-buffered saline (HBS) was added dropwise to form a precipitate that was added to the cells. The medium was replaced on the following day, and supernatants containing the pseudoparticles were harvested 48 h later, cleared by passage through 0.45-μm-pore-size filters, and used for luciferase infection assays.

Subgenomic replicon assay.

Huh7/Scr cells were seeded at a density of 5 × 104 cells/well in 24-well plates. On the following day, cells were transfected with electroporation with RNA of SGR-JFH1 according to the protocol described above. Four hours posttransfection, transfection medium was replaced by fresh medium, and compounds were added. Firefly luciferase activity was measured 48 h later as described above.

Cell-cell transmission assay.

Huh7/Scr cells (“donor cells”) were infected with the Jc1 virus at a multiplicity of infection (MOI) of ≥5 TCID50/cell. Twenty-four hours later, cells were trypsinized and counted, and 5 × 104 cells were cocultured in 24-well plates on coverslips with 5 × 104 Huh7.5/EGFP-NLS-IPS cells (“acceptor cells”). Cynaropicrin (10 μg/ml), grosheimol (20 μg/ml), erlotinib (10 μg/ml), and DMSO (0.1% [vol/vol]) were added simultaneously to the cells during seeding. Four hours later, medium was removed, and the cells were washed once with PBS and finally covered with fresh DMEM complete containing 1% low-melting-temperature agarose (Sigma-Aldrich, St. Louis, MO) and then cultivated for another 20 h prior to immunofluorescence analysis. To assess the efficiency of cell-cell infection, the proportion of infected cells was monitored by NS5A-specific indirect immunofluorescence (as described below). Quantification of Huh7.5/EGFP-NLS-IPS cells was done in 3 independent wells by taking 3 independent pictures of a different field of each well that contained at least 200 cells in total. Data are expressed as the percentage of DMSO-treated cells and represent mean values from the 3 independent fields of three biological replicates (±SEM).

Indirect immunofluorescence.

Twenty four hours postcocultivation of Huh7/Scr with Huh7.5/EGFP-NLS-IPS wells, medium containing 1% agarose was aspirated after being heated at 42°C for 10 min. Then cells were washed three times with PBS, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.5% Triton X-100 in PBS. Immunostaining of NS5A was performed by using the mouse monoclonal anti-NS5A antibody 9E-10 (kindly provided by Charles Rice, The Rockefeller University, USA) at a final concentration of 0.1 μg/ml in PBS supplemented with 5% bovine serum albumin (BSA). Bound primary antibodies were detected using goat anti-mouse antibodies conjugated to Alexa Fluor 568 (Invitrogen, Eugene, OR) at a dilution of 1:1,000 in PBS with 5% BSA. Finally, cells were washed 3× with PBS and once with water and mounted in Mowiol (Sigma-Aldrich, St. Louis, MO). Images were acquired on a Leica SP2 confocal microscope (Leica, Hicksville, NY).

Statistical analyses.

Mann-Whitney tests were performed in order to compare two unpaired groups using the Graphpad Prism 5.0 software. P values of <0.05, <0.01, and <0.001 were considered to indicate significant differences.

RESULTS

Identification of WEA-derived compounds with antiviral activity against HCV.

The phytochemical investigation of the WEA extract resulted in the isolation and characterization of a new sesquiterpene lactone (compound 1, designated “grosheimol”), as well as five additional known compounds (compounds 2 to 6 in Fig. 1). The complete data that describe the structural elucidation, absolute configuration, and quantitative determination of all compounds are available upon request. The anti-HCV activities of all six compounds were evaluated by using the highly permissive human hepatoma cell line Huh7/Scr and firefly luciferase reporter viruses based on the intragenotypic genotype 2a chimera Jc1 (Luc-Jc1 [17]) (Fig. 2A). Briefly, on the day of infection, Huh7/Scr cells were treated for 1 h with 10 or 20 μΜ each compound or dasatinib, a known HCV entry inhibitor (18), at the same concentrations. The total WEA extract was included in this experiment (at 100 and 200 μg/ml) in order to compare its anti-HCV capacity with the isolated compounds. Then medium containing the compounds was removed, and the cells were infected with fresh medium that contained Luc-Jc1 and compounds. At 4 h postinfection, the media were replaced again with fresh compound-containing media, and 72 h after infection the cells were assayed for firefly luciferase activity. Cell viability was monitored in parallel with an ATP assay (19). As shown in Fig. 2B, all 6 molecules and the WEA extract inhibited HCV cell entry and/or RNA replication. Compounds 1 and 2 (grosheimol and cynaropicrin) exhibited the greatest potency by inhibiting HCV more than 90% and 80%, respectively. For this, these compounds were characterized further with respect to their anti-HCV capacity.

FIG 1.

FIG 1

Chemical structures of the natural compounds isolated from WEA.

FIG 2.

FIG 2

Screening of WEA-derived compounds for antiviral activity against HCV. (A) Schematic drawing of the Luc-Jc1 reporter virus genome used for the screening. UTR, untranslated region; Fluc, firefly luciferase; EMCV, encephalomyocarditis virus; IRES, internal ribosomal entry site. (B) Ninety-six-well plates were seeded with Huh7/Scr cells at 1.2 × 104 cells/well 16 h prior to infections. The day of infections, cells were incubated with compounds for 1 h at the indicated concentrations. Then compound-containing media were removed, and cells were inoculated with Luc-Jc1 virus-compound preparations at the same concentrations. Finally, virus-compound preparations were replaced with fresh medium-compound preparations, and HCV infection efficiency was determined 72 h postinoculation using firefly luciferase assays. Cell viability was measured in parallel using an ATP assay. All data were plotted as percentages relative to DMSO for both infectivity and cell viability. Data are expressed as mean values ± SEM from four measurements of two biological replicates.

Cynaropicrin and grosheimol potently inhibit HCV.

To assess the potency of the isolated sesquiterpene lactones cynaropicrin and grosheimol against HCV, the Luc-Jc1 virus was used to estimate the half-maximal effective concentration (EC50), the half-maximal cytotoxic concentration (CC50), and the selectivity index (SI [CC50/EC50]) by performing a dose-response infection assay. Total WEA extract was included again in this assay in order to compare the potency of the isolated compounds versus that of the crude extract. Dasatinib served again as a positive control. The infection protocol and the compound incubation period were similar to those described above. As shown in Fig. 3A to D, the estimated EC50 for cynaropicrin was 1.3 μM (CC50, 16.9 μM; SI, 13.0), compared with an EC50 of 1.0 μM for grosheimol (CC50, 36.7 μM; SI, 36.7) and an EC50 of 1.5 μM for dasatinib (CC50, 20.3 μM; SI, 13.5) in this assay. As expected, estimated values for total extract (EC50, 299 μg/ml; CC50, 686 μg/ml; SI, 2.3) indicate a less potent formulation in comparison to the isolated compounds.

FIG 3.

FIG 3

Grosheimol and cynaropicrin inhibit HCV genotype 2a. Ninety-six-well plates were seeded with Huh7/Scr cells at 1.2 × 104 cells/well 16 h prior to infections. The day of infections, cells were treated with increasing concentrations of (A) cynaropicrin, (B) grosheimol, or (C) total artichoke-extract for 1 h. Then compound-containing media were removed, and cells were infected with a Luc-Jc1 virus-compound mixture at a multiplicity of infection (MOI) of 0.01 TCID50/cell. The tyrosine kinase inhibitor dasatinib, a known HCV entry inhibitor, served as a positive control (D). Virus-compound mixtures were replaced 4 h postinfection with fresh medium-compound mixture, and 72 h after infection, cells were assayed for firefly luciferase activity, and the mean relative light units (RLU) were plotted as percentages relative to DMSO for both infectivity and cell viability. The half-maximal effective concentration (EC50) and half-maximal cytotoxic concentration (CC50) were estimated by nonlinear regression of log inhibitor versus normalized response and used to calculate the selectivity index (SI) value. Data are expressed as mean values ± SEM from four measurements of two biological replicates.

Cynaropicrin and grosheimol inhibit HCVpp cell entry.

We then sought to determine at which step of the viral life cycle cynaropicrin and grosheimol exert their action: whether it was entry, replication/translation, or assembly/release. Different HCV-based systems exist that allowed us to dissect the different steps of the viral life cycle (20). To assess if cynaropicrin and grosheimol inhibit the entry step, we used the HCV pseudoparticle (HCVpp) system. HCVpp is a well-established system for the study of HCV entry and neutralization (21, 22). The HCVpp consist of lenti- or retroviral cores surrounded by an envelope displaying HCV E1E2 envelope glycoproteins. For consistency with the previous experiments, we used HCVpp that carry glycoproteins (genotype 2a, isolate J6CH) identical to those of the Luc-Jc1 virus. The day of infections, Huh7/Scr cells were treated for 1 h with high concentrations of the isolated compounds cynaropicrin (5 μM), grosheimol (20 μM), or the control, dasatinib (5 μΜ). Then cells were infected with the HCVpp-compound mixture at the same concentration. Six hours postinfection, HCVpp-containing medium was replaced with fresh medium-compound mixture, and 72 h after infection, cells were assayed for renilla luciferase activity. HCVpp were inhibited in the presence of cynaropicrin (∼70%) or grosheimol (∼40%) (Fig. 4A, left). These results indicate that both compounds act at least at the entry level. The positive control, dasatinib, also reduced infectivity levels of the HCVpp (∼60%) as expected. Consistent with the results in Fig. 2, neither grosheimol nor cynaropictin showed cytotoxicity at the concentrations used in this assay (Fig. 4A, right). HCVpp are lentiviral particles that harbor on their surface the HCV glycoproteins. Early entry steps of HCV are recapitulated by these particles, but late entry steps are dependent on HIV replication mechanisms. To discriminate between an effect of cynaropicrin and grosheimol on HCV envelope protein functions and the process leading to lentiviral integration and expression, we performed the exact same experiment using pseudoparticles that harbor on their surface the glycoprotein of the vesicular stomatitis virus (VSV-G). As shown in Fig. 4B, only cynaropicrin showed an ∼30% inhibition in entry or expression of these particles. According to this result, we cannot exclude a low inhibitory capacity of cynaropicrin on entry of VSV-G or expression of HIV lentiviral particles. However, the results presented in Fig. 4A in combination with the results in previous figures point out the strong inhibitory capacity of this compound against HCV.

FIG 4.

FIG 4

Cynaropicrin and grosheimol inhibit HCVpp cell entry. Ninety-six-well plates were seeded with Huh7/Scr cells at 1.2 × 104 cells/well 16 h prior to infections. The day of infections, cells were treated with 5 μΜ cynaropicrin, 20 μΜ grosheimol, or 5 μΜ dasatinib for 1 h. Then compound-containing media were removed, and cells were infected with HCV pseudoparticle (HCVpp)-compound mixture at the same concentration (A) or pseudotypes carrying on their surface VSV-G (B). HCVpp were carrying identical glycoproteins (genotype 2a, isolate J6CF) to the Luc-Jc1 viruses. Pseudotype-compound mixtures were replaced 6 h postinfection with fresh medium-compound mixture, and 72 h after infection, cells were assayed for renilla luciferase activity, and the mean relative light units (RLU) were plotted as percentages relative to DMSO for both infectivity and cell viability. Data are expressed as mean values ± SEM from four measurements of two biological replicates. *, P < 0.05; **, P < 0.01.

Cynaropicrin and grosheimol inhibit early steps of HCVcc entry.

HCV entry is a multistep process that involves viral proteins and several cellular receptors. Cynaropicrin- and grosheimol-mediated inhibition of HCVpp could be a result of either a block in virus-cell binding, virus uptake, or viral delivery into the cytoplasm. To characterize at which step of HCV entry grosheimol and cynaropicrin act, we investigated the inhibitory capacities of the different compounds when administered at different intervals during the early phase of infection (Fig. 5). We used again a high-titer reporter virus preparation of Luc-Jc1 and incubated it in the presence or absence of inhibitors with Huh7/Scr cells for 1 h at 4°C. Under these conditions, virus attaches to the cells but does not efficiently enter, thus permitting a rather synchronous infection when the inoculum is removed and cells are shifted to 37°C. Therefore, subsequent to virus attachment, unbound virus was washed away, cells were transferred to 37°C, and inhibitors were added either directly or 30, 60, 90, 120, or 240 min later for an interval of 4 h, as indicated in Fig. 5A. Heparin, which is known to inhibit HCV attachment (17), dasatinib, which is known to inhibit late steps in the entry process (18), and chlorpromazine, which inhibits fusion by causing clathrin lattices to assemble on endosomal membranes and at the same time prevents the assembly of coated pits at the plasma membrane (23), were used as controls. Under these conditions, we found that heparin inhibited the infection only when it was present during virus binding, as expected (Fig. 5B). Interestingly, both grosheimol and cynaropicrin inhibited virus binding and when added to cells at the point of temperature shift from 4°C to 37°C, clearly indicating that these compounds play an inhibitory role during the early steps of HCV entry. Dasatinib and chrorpromazine exhibited strong inhibition when added early during infection, while they kept their inhibitory capacity up to 120 min postshift at 37°C, confirming their role in late HCV entry events.

FIG 5.

FIG 5

Kinetics of inhibitory activity exerted by cynaropicrin and grosheimol. (A) Schematic drawing of the experimental setup of the experiment depicted in panel B. Inhibition of Luc-Jc1 entry into Huh7/Scr cells by heparin (100 μg/ml), cynaropicrin (5 μΜ), grosheimol (20 μΜ), dasatinib (5 μΜ), and chlorpromazine (5 μg/ml) was compared using seven different experimental protocols (indicated by roman numerals i through vii). Virus binding to target cells was performed for 1 h at 4°C in the absence (ii to vii) or presence (i) of compounds. Subsequently, cells were washed with PBS and shifted to 37°C to allow entry to proceed. Depending on the protocol, inhibitors were added directly or 30, 60, 90, 120, or 240 min thereafter (ii, iii, iv, v, vi, and vii, respectively). Dotted lines indicate the time interval during which an inhibitor was present; black arrows indicate the addition and removal of virus inoculum (B). The efficiency of infections using the protocols described in panel A was determined by luciferase assays 72 h postinfection. The mean relative light units (RLU) were plotted as percentages relative to DMSO. Data are expressed as mean values ± SEM from four measurements of two biological replicates.

Cynaropicrin and grosheimol act directly on HCV particles.

According to the previous results, grosheimol and cynaropicrin inhibit HCV during the early events of its entry. However, these compounds could be either acting directly on the viral particle, mediating an effect on the host cell, or both. In order to address this issue, we performed time-of-addition experiments in which either the cells or the virus was preincubated for 1 h with compounds prior to virus inoculation, a virus-compound mixture was directly added to the cells, or compounds were added after 4 h of virus inoculation (Fig. 6, left). Both grosheimol and cynaropicrin interfered with HCV infection when viruses were incubated with the compounds and then compounds were present during virus inoculation (Fig. 6, right). Preexposure of the cells to the compounds or addition 4 h after infection did not result in HCV inhibition. These data corroborate the previous results and point to a direct HCV particle inhibition mechanism.

FIG 6.

FIG 6

Mode-of-action analysis of cynaropicrin and grosheimol by time-of-addition experiments. Ninety-six-well plates were seeded with Huh7/Scr cells at 1.2 × 104 cells/well 16 h prior to infections. The day of infections, cells were inoculated with Luc-Jc1 reporter viruses prepared in the absence of drugs. Cynaropicrin (5 μM) or grosheimol (20 μM) was added to the cells only before inoculation (black), was added to viruses and preincubated with them at 37°C prior to inoculation (stripes), or was added selectively directly after inoculation (white), as schematically depicted to the left. Infectivity was determined 72 h later by firefly luciferase assays, and the mean relative light units (RLU) were plotted as percentages relative to DMSO for both infectivity and cell viability. Data are expressed as mean values ± SEM from four measurements of two biological replicates.

Cynaropicrin and grosheimol neither inhibit HCV RNA replication and/or translation nor HCV particle production.

To investigate the impact of cynaropicrin and grosheimol in HCV RNA translation and/or replication, we transfected Huh7/Scr cells with a subgenomic JFH1 luciferase replicon (SGR-JFH1) (Fig. 7A, top) (12). Subgenomic replicons are autonomously replicating molecules that lack structural proteins. A reporter gene (firefly luciferase) is expressed via the HCV IRES followed by the encephalomyocarditis virus (EMCV) IRES, which drives the expression of nonstructural proteins (NS3 to NS5B). As a result, the viral RNA is translated and replicates but does not encapsidate or produce new virions. Briefly, Huh7/Scr cells were electroporated with the SGR-JFH1 RNA. Cynaropicrin or grosheimol was added after 4 h, and luciferase activity was assayed after 48 h. Changes in luciferase levels correlate with levels of HCV replication and/or translation. A 2′-modified nucleoside analog (2′-C-methyladenosine, 2′-C-Met) (24) was used as a positive control. As shown in Fig. 7A (bottom), none of the tested compounds inhibited HCV RNA replication and/or translation, while the control compound exerted a strong inhibitory effect in this assay, suggesting that the WEA-isolated compounds do not act at these viral life cycle steps.

FIG 7.

FIG 7

Neither grosheimol nor cynaropicrin inhibits HCV translation/replication or particle production and egress. (A) Huh7/Scr cells were transfected by electroporation with the subgenomic firefly luciferase replicon depicted at the top. Ninety-six-well plates were seeded with electroporated cells at a concentration of 1.2 × 104 cells/well. Compounds were added 4 h posttransfection at a final concentration of 5 μM or 20 μM (for cynaropicrin and 2′-C-Met or grosheimol, respectively), and the levels of HCV RNA translation/replication were quantified by firefly luciferase assays 72 h posttransfection. The 2′-modified nucleoside analog 2′-C-methyladenosine (2′-C-Met) (24) was used as a positive control. Data are expressed as mean values ± SEM from four measurements of two biological replicates. (B) A schematic representation of the experimental procedure is depicted at the top. Huh7/Scr cells were electroporated with Luc-Jc1 RNA and replicate tissue culture plates were seeded with them. Artichoke-derived compounds and the known HCV particle production inhibitor U0126 were added to the medium at 40 h postelectroporation at the given concentrations. Two hours later, cells were washed 3 times with PBS to remove the compounds and fed for 6 h with fresh medium. Finally, at 48 h postelectroporation, supernatant was harvested and cells were lysed. HCV RNA replication in cells was measured by using a firefly luciferase reporter assay (top panel). The release of infectious particles was determined by inoculation of naive cells with the collected culture fluids and determination of firefly luciferase activity in naive cells 72 h after inoculation (bottom panel). Data are expressed as mean values ± SEM from four measurements of two biological replicates.

Cynaropicrin and grosheimol do not inhibit HCV particle production and egress.

To investigate the potential role of the isolated sesquiterpene lactones in HCV particle production, we adapted and slightly modified an HCV particle production assay described by Menzel et al. (25). Briefly, as shown in Fig. 7B, top, Huh/Scr cells were electroporated with Luc-Jc1 virus and cultured for 40 h. At this time point, cells were extensively washed with PBS and fed with fresh medium containing cynaropicrin or grosheimol for 2 h. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibitor U0126, which has been shown to inhibit HCV particle production, served as the positive control. After this 2-h compound treatment, cells were washed again extensively and fed with fresh medium for 6 h to allow particle production post-compound treatment. Finally, the treated medium was collected, clarified from cells by centrifugation, and tested for particle production in naive Huh7/Scr cells, while the effect of the compounds on HCV replication/translation and their cytotoxicity was measured in the electroporated cells by luciferase assays. As shown at the bottom of Fig. 7B, neither cynaropicrin nor grosheimol inhibited HCV particle production in this assay, in contrast to U0126, which showed inhibition without any effect on replication/translation. These data narrow the effects of cynaropicrin and grosheimol at the entry level.

Cynaropicrin and grosheimol are active against all major HCV genotypes.

HCV isolates have been classified into seven major genotypes (17), differing in their nucleotide sequence by around 30%, and a number of subtypes (a, b, etc.) with ∼20% sequence divergence (26). HCV treatment efficacy is influenced by viral genotype—and even subtype in the case of HCV genotype 1 infection—and consequently, treatment decisions are made taking this information into consideration (27). All previous experiments in this study were performed using viruses derived from genotype 2a. In order to determine if WEA-isolated compounds are also active against the other HCV genotypes, we used chimeric JFH-1-based reporter virus constructs, carrying renilla luciferase inserted at the NS5A gene and structural proteins from all major HCV genotypes and the most relevant genotype 1 subtypes: 1a (isolate TN), 1b (isolate J4), 2b (isolate J8), 3a (isolate S52), 4a (isolate ED43), 5a (isolate SA13), 6a (isolate HK6a), and 7a (isolate QC69) (Fig. 8A) (15). Similar dose-response inhibition experiments as described above were performed in order to evaluate the potency of cynaropicrin and grosheimol against multiple HCV genotypes. Importantly, infectivity was reduced under this treatment condition for all different genotypes (Fig. 8B), indicating that WEA-isolated compounds inhibit HCV infection independently of viral genotype or subtype. The estimated EC50, CC50, and SI for all major HCV genotypes are presented in Table 1.

FIG 8.

FIG 8

Grosheimol and cynaropicrin inhibit HCV of different genotypes. (A) Schematic drawing of the renilla luciferase-carrying reporter virus genomes used for this screening. (B) Ninety-six-well plates were seeded with Huh7/Scr cells at 1.2 × 104 cells/well 16 h prior to infections. The day of infections, cells were preincubated for 1 h with compounds at the given concentrations. Then compound-containing media were removed, and cells were inoculated with genotype 1 to 7 virus-compound preparations at the same concentrations. Finally, virus-compound preparations were replaced with fresh medium (containing compounds) 4 h postinoculation, and HCV infection efficiency was determined 72 h postinoculation using renilla luciferase assays and expressed as a percentage of the DMSO control. Data are expressed as mean values ± SEM from four measurements of two biological replicates.

TABLE 1.

Antiviral activities of grosheimol and cynaropicrin across all major HCV genotypes

HCV genotype (isolate) Cynaropicrin
Grosheimol
EC50 (μM) CC50 (μM) SI EC50 (μM) CC50 (μM) SI
1a (TN) 0.4 16.8 40.5 2.7 76.4 28.3
1b (J4) 1.1 15.7 14.3 4.5 89.0 19.8
2b (J8) 0.7 19.4 27.7 7.2 103.3 14.3
3a (S52) 0.7 18.1 25.6 7.3 87.6 12.0
4a (ED43) 0.7 13.8 19.7 8.7 85.9 9.9
5a (SA13) 0.8 14.4 18.0 14.0 90.8 6.5
6a (HK6a) 0.7 17.8 25.4 6.3 92.2 14.6
7a (QC69) 1.4 15.1 10.8 5.7 86.7 15.2

Impact of cynaropicrin and grosheimol in HCV cell-cell transmission.

HCV is transmitted between hepatocytes via classical cell entry using cell-free diffusion, but it also uses direct cell-cell transfer to infect neighboring cells (28). All previously described assays where cynaropicrin and grosheimol inhibited HCV entry were based on cell-free diffusion transmission. To investigate the capacity of these compounds to inhibit HCV transmission in a cell-cell assay, we developed a cell-cell HCV inhibition assay based on that published by Lupberger et al. (18). In this assay (Fig. 9A), a coculture of 2 different cell types takes place: Huh7/Scr cells infected with the Jc1 virus act as HCV donor cells, while Huh7.5/EGFP-NLS-IPS cells act as acceptor cells (10). In the latter cells, upon HCV infection, the NS3-4A protease cleaves the IPS-1 protein (i.e., IFN-β promoter stimulator protein 1), which normally localizes to mitochondria, and subsequently the enhanced fluorescent protein (EGFP) localizes to the nucleus due to its fusion to a nuclear localization signal (NLS). To avoid cell-free virus infection, the coculture is overlaid with agarose. As shown in Fig. 9B and C, cynaropicrin and grosheimol efficiently inhibited cell-cell transmission, while the solvent (DMSO) did not prevent HCV infection of the Huh7.5/EGFP-NLS-IPS cells. The reversible tyrosine kinase inhibitor erlotinib, which has been also shown to inhibit HCV cell-cell transmission (18), was used as a positive control.

FIG 9.

FIG 9

Grosheimol and cynaropicrin inhibit HCV cell-cell transmission. (A) Cell culture model system for analysis of HCV cell-cell transmission. In this assay, Huh7/Scr cells are infected with the Jc1 virus and function as HCV “donor” cells containing replicating HCVcc (stained by an anti-NS5A antibody and indicated in red) and cocultured with HCV Huh7.5/EGFP-NLS-IPS “acceptor” cells for 24 h. In the latter cells, upon HCV infection, the NS3-4A protease cleaves the IPS-1 protein, which normally localizes to mitochondria, and subsequently the EGFP (indicated in green) localizes to the nucleus due to its fusion to a nuclear localization signal (NLS). Fluorescence microscopy is used to quantitate infected HCV+ Huh7.5/EGFP-NLS-IPS “acceptor” cells (indicated by red and green), which are infected via cell-cell transmission and stained with anti-NS5A antibody. (B) Huh7/Scr cells were infected with Jc1 virus at an MOI of ∼10 TCID50/cell, and 20 h later, 24-well plates were seeded with them at 5 × 104 cells/well. Simultaneously, the same 24-well plates were seeded with Huh7.5/EGFP-NLS-IPS cells (coculture), and cynaropicrin (10 μM), grosheimol (20 μM), or erlotinib (10 μM) was added to the wells. The addition of the compounds' solvent (DMSO) served as a negative control. Four hours later, medium was removed, and cells were overlaid with 1% agarose with fresh compounds. Twenty-four hours later, the HCV infection was analyzed by immunofluorescence of NS5A staining. Quantification of Huh7.5/EGFP-NLS-IPS cells was done in 3 independent wells by taking 3 independent pictures of different fields of each well that contained at least 200 cells in total. Data are expressed as percentages of DMSO-treated cells and represent mean values ± SEM from the 3 independent fields of three biological replicates. ***, P < 0.001. (C) Immunofluorescence analysis of NS5A protein (red) in coculture of Huh7/Scr Jc1-infected cells with Huh7/EGFP-NLS-IPS, 24 h postcoculture and treated with erlotinib (10 μΜ), cynaropicrin (10 μΜ), or grosheimol (20 μΜ) or mock treated (DMSO). Magnification, 63×.

DISCUSSION

The present study demonstrates an anti-HCV activity of two isolated and characterized compounds from aqueous leaf extracts of the wild Egyptian artichoke (WEA). The first previously unknown compound was designated grosheimol, while the second was identified as cynaropicrin. Both compounds belong to the class of sesquiterpene lactones and exhibited pan-genotypic anti-HCV activity with EC50s in the low micromolar range. They inhibit HCV entry into target cells and thus represent interesting hit compounds for further drug development. These findings support the efforts to use folk medicinal plants as a source in the search for pharmacologically active compounds.

A number of promising natural products with anti-HCV activities have been described (for recent reviews, see references 2932). They were of different origins and chemical structures and exerted their antiviral effects at different levels within the virus life cycle. Among these were several sesquiterpene lactones derived from parthenolide, an active component of feverfew that has been used against fever and migraine (33). An analysis of the structure-activity relationship of parthenolide and various analogs as inhibitors of HCV replication has been performed with a subgenomic HCV replicon system and has shown that the α-methylene-γ-lactone moiety was important for maximal antiviral activity (33). Albeit grosheimol and cynaropicrin inhibit HCV at the level of viral entry into target cells and not replication, they interestingly contain the same structural element as parthenolide. This suggests a functional meaning in the context of the viral entry process as well and deserves further investigation.

HCV entry into hepatocytes is a complex, multistep well-orchestrated process that involves the HCV envelope glycoproteins E1 and E2 as well as several host factors (34). HCV entry is an attractive target for antiviral drug development because it offers the advantage of combating the infection at its initial steps before virus progeny are generated that can persist by ongoing replication. Presently, several entry inhibitors have been described, and their modes of action have been determined in mouse models or in cell culture (35). Among them, a plant-derived flavonoid (36) has been shown to inhibit the entry of all major HCV genotypes in vitro, while the green tea polyphenol epigallocatechin-3-gallate has been shown to inhibit primarily HCV entry (37, 38) and secondarily HCV replication (39). Nonetheless, most entry inhibitors are in preclinical development, and only 2 of them are presently in clinical phase I/IIa of development. These are the ITX5061 inhibitor of the scavenger receptor class B type I and erlotinib, a known compound that inhibits the epidermal growth factor alpha receptor. Very recently, the coadministration of the anti-SRBI mAb1671 with the DAA ciluprevir (protease inhibitor) in human liver-grafted mice offered the first preclinical in vivo evidence that addition of an entry inhibitor to an anti-HCV DAA regimen restricts the breakthrough of DAA-resistant viruses (36). Thus, they may present in the future an appropriate solution for (i) prevention of the liver graft infections that are universally observed when seropositive patients are not treated prior to liver transplantation and for (ii) treatment in combination with DAAs of difficult-to-treat patients like those with liver cirrhosis and hepatocellular carcinoma.

Our data indicate that cynaropicrin and grosheimol prevent HCV life cycle steps that include the virus binding and early entry processes. Importantly, both compounds inhibited HCV only when viruses were preincubated with the compounds but not the cells. These findings strongly suggest that both compounds act directly on viral particles and may prevent virus-receptor interactions. Considering the antiviral activities of grosheimol and cynaropicrin against all HCV genotypes, these compounds present very attractive pan-genotypic anti-HCV natural products. Nevertheless, further studies regarding their toxicity are required. In our studies, although the selectivity of both compounds was comparable to that obtained with the anticancer drug dasatinib, which is a Bcr-Abl tyrosine kinase inhibitor, full inhibition of HCV infectivity was achieved at cytotoxic concentrations. Additional studies with derivatives of cynaropicrin and grosheimol might help to overcome these difficulties in the future.

Medicinal food plants, including artichokes, are in widespread use—and sometimes have been in use for centuries (4044). Their medical benefit is often anecdotal and does not always hold up to strict scientific criteria (45). Nonetheless, numerous interesting compounds with a broad range of bioactivities have been isolated from them, and they present interesting lead compounds for further development into specific pharmaceuticals. A good example is cynaropicrin, which besides its anti-HCV activity, described here for the first time, is active against the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (sleeping sickness) (46). It mediates the effect by targeting the trypanothione redox system of the parasite (47). Additionally, it has been shown that cynaropicrin exhibits inhibitory effects on the production of tumor necrosis factor alpha, which is a proinflammatory cytokine (48). Furthermore, its antihyperlipidemic (49), anticancer (50) and antiphotoaging (51) activities indicate that cynaropicrin has a diverse pharmaceutic potential. Because (i) semisynthetic derivatives of cynaropicrin with improved bioactivity properties can be generated with relative ease (52) and (ii) the genes encoding its biosynthesis in plants are starting to be characterized (53) and thus may become available for engineering of microorganisms for cynaropicrin biosynthesis, there seem to be a number of efficient production options to generate affordable pharmaceutical products.

In conclusion, cynaropicrin and grosheimol are two natural products within aqueous WEA that have potent anti-HCV activities. Their chemical properties and ease of isolation and modification make them interesting candidates for further development into early step HCV inhibitors.

ACKNOWLEDGMENTS

We thank Päivi Joensuu (Mass Laboratory, Department of Chemistry, University of Oulu) and Ulrich Bergmann (Department of Biochemistry, University of Oulu) for technical assistance.

Funding Statement

This work was supported by FEDER and the Spanish Ministry of Economy and Competitiveness through grants BFU 2013-44629-R and SAF2013-46077-R and the Maria de Maeztu Programme for Units of Excellence in R&D (MDM-2014-0370).

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