Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice.

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Eremoxylarins D-J, Antibacterial Eremophilane Sesquiterpenes Discovered from an Endolichenic Strain of Xylaria hypoxylon.

An endolichenic strain of the Ascomycetaceous Xylaria hypoxylon, cultivated alone or in coculture with another endolichenic fungus Dendrothyrium variisporum, produced seven new bioactive eremophilane sesquiterpenes eremoxylarins D-J (1-7). The isolated compounds disclosed a high similarity with the eremophilane core of the bioactive integric acid, and structures were elucidated by 1D and 2D NMR spectra and electronic circular dichroism (ECD) analyses. Eremoxylarins D, F, G, and I showed a selective activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus with minimum inhibitory concentration (MIC) values between 0.39 and 12.5 µg/mL. Eremoxylarin I, the most antibacterial active sesquiterpene, was also active against HCoV-229E at a concentration nontoxic to the hepatoma Huh-7 cell line with an 50% inhibitory concentration (IC50) of 18.1 µM and a 50% cytotoxic concentration (CC50) of 46.6 µM.

Discovery of Anti-Coronavirus Cinnamoyl Triterpenoids Isolated from Hippophae rhamnoides during a Screening of Halophytes from the North Sea and Channel Coasts in Northern France.

The limited availability of antiviral therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spurred the search for novel antiviral drugs. Here, we investigated the potential antiviral properties of plants adapted to high-salt environments collected in the north of France. Twenty-five crude methanolic extracts obtained from twenty-two plant species were evaluated for their cytotoxicity and antiviral effectiveness against coronaviruses HCoV-229E and SARS-CoV-2. Then, a bioguided fractionation approach was employed. The most active crude methanolic extracts were partitioned into three different sub-extracts. Notably, the dichloromethane sub-extract of the whole plant Hippophae rhamnoides L. demonstrated the highest antiviral activity against both viruses. Its chemical composition was evaluated by ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) and then it was fractionated by centrifugal partition chromatography (CPC). Six cinnamoyl triterpenoid compounds were isolated from the three most active fractions by preparative high-performance liquid chromatography (HPLC) and identified by high resolution MS (HR-MS) and mono- and bi-dimensional nuclear magnetic resonance (NMR). Specifically, these compounds were identified as 2-O-trans-p-coumaroyl-maslinic acid, 3ß-hydroxy-2α-trans-p-coumaryloxy-urs-12-en-28-oic acid, 3ß-hydroxy-2α-cis-p-coumaryloxy-urs-12-en-28-oic acid, 3-O-trans-caffeoyl oleanolic acid, a mixture of 3-O-trans-caffeoyl oleanolic acid/3-O-cis-caffeoyl oleanolic acid (70/30), and 3-O-trans-p-coumaroyl oleanolic acid. Infection tests demonstrated a dose-dependent inhibition of these triterpenes against HCoV-229E and SARS-CoV-2. Notably, cinnamoyl oleanolic acids displayed activity against both SARS-CoV-2 and HCoV-229E. Our findings suggest that Hippophae rhamnoides could represent a source of potential antiviral agents against coronaviruses.

New Phenolic Lipids from the Leaves of Clausena harmandiana Inhibit SARS-CoV-2 Entry into Host Cells.

Induced by the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic underlined the clear need for antivirals against coronaviruses. In an effort to identify new inhibitors of SARS-CoV-2, a screening of 824 extracts prepared from various parts of 400 plant species belonging to the Rutaceae and Annonaceae families was conducted using a cell-based HCoV-229E inhibition assay. Due to its significant activity, the ethyl acetate extract of the leaves of Clausena harmandiana was selected for further chemical and biological investigations. Mass spectrometry-guided fractionation afforded three undescribed phenolic lipids (1-3), whose structures were determined via spectroscopic analysis. The absolute configurations of 1 and 2 were determined by analyzing Mosher ester derivatives. The antiviral activity against SARS-CoV-2 was subsequently shown, with IC50 values of 0.20 and 0.05 µM for 2 and 3, respectively. The mechanism of action was further assessed, showing that both 2 and 3 are inhibitors of coronavirus entry by acting directly on the viral particle. Phenolic lipids from Clausena harmandiana might be a source of new antiviral agents against human coronaviruses.

Luteolin Isolated from Juncus acutus L., a Potential Remedy for Human Coronavirus 229E.

The COVID-19 pandemic, caused by SARS-CoV-2, addressed the lack of specific antiviral drugs against coronaviruses. In this study, bioguided fractionation performed on both ethyl acetate and aqueous sub-extracts of Juncus acutus stems led to identifying luteolin as a highly active antiviral molecule against human coronavirus HCoV-229E. The apolar sub-extract (CH2Cl2) containing phenanthrene derivatives did not show antiviral activity against this coronavirus. Infection tests on Huh-7 cells, expressing or not the cellular protease TMPRSS2, using luciferase reporter virus HCoV-229E-Luc showed that luteolin exhibited a dose-dependent inhibition of infection. Respective IC50 values of 1.77 µM and 1.95 µM were determined. Under its glycosylated form (luteolin-7-O-glucoside), luteolin was inactive against HCoV-229E. Time of addition assay showed that utmost anti-HCoV-229E activity of luteolin was achieved when added at the post-inoculation step, indicating that luteolin acts as an inhibitor of the replication step of HCoV-229E. Unfortunately, no obvious antiviral activity for luteolin was found against SARS-CoV-2 and MERS-CoV in this study. In conclusion, luteolin isolated from Juncus acutus is a new inhibitor of alphacoronavirus HCoV-229E.

A Photoactivable Natural Product with Broad Antiviral Activity against Enveloped Viruses, Including Highly Pathogenic Coronaviruses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.

Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release.

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.

Dehydrojuncusol, a Natural Phenanthrene Compound Extracted from Juncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication.

Recent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCE Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes.

Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells. The relevance of cell polarity in the HCV life cycle has been addressed only in distantly related models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins typical of columnar polarized epithelia, which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used, and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on a semipermeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture-derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density were secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle.IMPORTANCE Hepatitis C is a major health burden, with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distantly related models and remains unclear. Hepatocyte organization is complex, with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with unique apical and basolateral domains was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower-density viral particles were produced under conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection.

Identification of a New Benzimidazole Derivative as an Antiviral against Hepatitis C Virus.

UNLABELLED: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle. IMPORTANCE: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

Identification of class II ADP-ribosylation factors as cellular factors required for hepatitis C virus replication.

GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G-proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf-guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA-mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.

Characterization of hepatitis C virus interaction with heparan sulfate proteoglycans.

UNLABELLED: Hepatitis C virus (HCV) entry involves binding to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction in the context of a viral infection. Patient sera and monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pulldown assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with the virion-heparin interaction, strongly suggesting that at the virion surface, HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pulldown experiments, and inhibition experiments with anti-apolipoprotein E antibodies indicated that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of the HS structural determinants required for HCV infection by silencing of the enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation is required for HCV infection and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures. IMPORTANCE: Hepatitis C is a global health problem. Hepatitis C virus (HCV) infects approximately 130 million individuals worldwide, with the majority of cases remaining undiagnosed and untreated. In most infected individuals, the virus evades the immune system and establishes a chronic infection. As a consequence, hepatitis C is the leading cause of cirrhosis, end-stage liver disease, hepatocellular carcinoma, and liver transplantation. Virus infection is initiated by entry of the virus into the host cell. In this study, we provide new insights into the viral and cellular determinants involved in the first step of HCV entry, the binding of the virus to host cells. We show that apolipoprotein E is likely responsible for virus binding to heparan sulfate and that N- and 6-O-sulfation of the heparan sulfate proteoglycans is required for HCV infection. In addition, the minimal HS length unit required for HCV infection is a decasaccharide.

Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action.

UNLABELLED: Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface. IMPORTANCE: In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors.

Khaya grandifoliola C.DC: a potential source of active ingredients against hepatitis C virus in vitro.

In this study, we examined the antiviral properties of Khaya grandifoliola C.DC (Meliaceae) on the hepatitis C virus (HCV) life cycle in vitro and identified some of the chemical constituents contained in the fraction with the most antiviral activity. Dried bark powder was extracted by maceration in a methylene chloride/methanol (MCM) system (50:50; v/v) and separated on silica gel by flash chromatography. Infection and replication rates in Huh-7 cells were investigated by luciferase reporter assay and indirect immunofluorescence assay using subgenomic replicons, HCV pseudotyped particles, and cell-culture-derived HCV (HCVcc), respectively. Cell viability was assessed by MTT assay, and cellular gene expression was analysed by qRT-PCR. The chemical composition of the fraction with the most antiviral activity was analysed by coupled gas chromatography and mass spectrometry (GC-MS). Five fractions of different polarities (F0-F100) were obtained from the MCM extract. One fraction (KgF25) showed the strongest antiviral effect on LucUbiNeoET replicons at nontoxic concentrations. Tested at 100 µg/mL, KgF25 had a high inhibitory effect on HCV replication, comparable to that of 0.01 µM daclatasvir or 1 µM telaprevir. This fraction also inhibited HCVcc infection by mostly targeting the entry step. KgF25 inhibited HCV entry in a pan-genotypic manner by directly inactivating free viral particles. Its antiviral effects were mediated by the transcriptional upregulation of the haem oxygenase-1 gene and interferon antiviral response. Three constituents, namely, benzene, 1,1'-(oxydiethylidene)bis (1), carbamic acid, (4-methylphenyl)-, 1-phenyl (2), and 6-phenyl, 4-(1'-oxyethylphenyl) hexene (3), were identified from the active fraction KgF25 by GC-MS. Khaya grandifoliola contains ingredients capable of acting on different steps of the HCV life cycle.

Identification of conserved residues in hepatitis C virus envelope glycoprotein E2 that modulate virus dependence on CD81 and SRB1 entry factors.

UNLABELLED: In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies. IMPORTANCE: Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

Structure-activity studies of (-)-epigallocatechin gallate derivatives as HCV entry inhibitors.

Preventing viral entry into cells is a recognized approach for HIV therapy and has attracted attention for use against the hepatitis C virus (HCV). Recent reports described the activity of (-)-epigallocatechin gallate (EGCG) as an inhibitor of HCV entry with modest potency. EGCG is a polyphenolic natural product with a wide range of biological activity and unfavorable pharmaceutical properties. In an attempt to identify more drug-like EGCG derivatives with improved efficacy as HCV entry inhibitors, we initiated structure-activity investigations using semi-synthetic and synthetic EGCG analogs. The data show that there are multiple regions in the EGCG structure that contribute to activity. The gallate ester portion of the molecule appears to be of particular importance as a 3,4-difluoro analog of EGCG enhanced potency. This derivative and other active compounds were shown not to be cytotoxic in Huh-7 cell culture. These data suggest that more potent, non-cytotoxic EGCG analogs can be prepared in an attempt to identify more drug-like candidates to treat HCV infection by this mechanism.

Antibacterial and anti-coronavirus investigation of selected Senegalese plant species according to an ethnobotanical survey.

ETHNOPHARMACOLOGICAL RELEVANCE: In Senegal, upper and lower respiratory tract infections constitute a real health problem. To manage these disorders, most people rely on the use of local medicinal plants. This is particularly the case for species belonging to the botanical families, Combretaceae, Fabaceae, Myrtaceae and Rubiaceae, which are widely used to treat various respiratory problems such as colds, flu, rhinitis, sinusitis, otitis, angina, bronchitis, bronchiolitis and also pneumonia. AIM OF THE STUDY: The aim of this study was to identify medicinal plants traditionally used for the management of infectious diseases, in particular those of the respiratory tract. On the basis of these ethnopharmacological uses, this study made it possible to highlight the antibacterial, antiviral and cytotoxic activities of selected plant species. MATERIALS AND METHODS: An ethnobotanical survey was conducted in Senegal among informants, including herbalists, traditional healers, and households, using medicinal plants in the management of infectious diseases, with a focus on respiratory tract infections. The most cited plant species were evaluated in vitro on a panel of 18 human pathogenic bacteria may be involved in respiratory infections and against the human coronavirus HCoV-229E in Huh-7 cells. The antiviral activity of the most active extracts against HCoV-229E was also evaluated on COVID-19 causing agent, SARS-CoV-2 in Vero-81 cells. In parallel, cytotoxic activities were evaluated on Huh-7 cells. RESULTS: A total of 127 informants, including 100 men (78.74%) and 27 women (21.26%) participated in this study. The ethnobotanical survey led to the inventory of 41 plant species belonging to 19 botanical families used by herbalists and/or traditional healers and some households to treat infectious diseases, with a specific focus on upper respiratory tract disorders. Among the 41 plant species, the most frequently mentioned in the survey were Guiera senegalensis J.F. Gmel. (95.2%), Combretum glutinosum Perr. Ex DC. (93.9%) and Eucalyptus spp. (82.8%). Combretaceae (30.2%) represented the most cited botanical family with six species, followed by Fabaceae (29.3%, 12 species). A total of 33 crude methanolic extracts of the 24 plant species selected for their number of citations were evaluated in vitro for their antimicrobial and cytotoxic activities. Guiera senegalensis, Combretum glutinosum, Vachellia nilotica subsp. tomentosa (Benth.) Kyal. & Boatwr, Eucalyptus camaldulensis Dehnh., and Terminalia avicennioides Guill. & Perr., showed antibacterial activities. The most active plants against HCoV-229E were: Ficus sycomorus L., Mitragyna inermis (Willd.) Kuntze, Pterocarpus erinaceus Poir., and Spermacoce verticillata L. One of these plants, Mitragyna inermis, was also active against SARS-CoV-2. CONCLUSION: This work confirmed the anti-infective properties of plant species traditionally used in Senegal. Overall, the most frequently cited plant species showed the best antibacterial activities. Moreover, some of the selected plant species could be considered as a potential source for the management of coronavirus infections. This new scientific data justified the use of these plants in the management of some infectious pathologies, especially those of the respiratory tract.

Antiviral miliusanes and isolation of an unprecedented miliusane dimer from Miliusa balansae.

In an extensive screening endeavor for anti-coronaviral compounds, we examined 824 tropical plant extracts from the Annonaceae and Rutaceae families. The screening identified an ethyl acetate extract from the aerial parts of Miliusa balansae for its potent inhibitory activity against Human coronavirus HCoV-229E. Subsequent bioassay-guided fractionation of this extract revealed two unreported miliusanes including a complex dimeric structure and seven known compounds, comprising miliusane XXXVI, (+)-miliusol, bistyryls, styryl-pyranones, and the flavonoid rhamnetin. The absolute configuration of the new dimeric miliusane was determined by X-ray crystallography and a putative biogenetic origin was proposed. Investigation of the antiviral effect of these nine phytochemicals within HCoV-229E-infected Huh-7 cells showed that (+)-miliusol and miliusane XXXVI exert antiviral activity at non-cytotoxic concentrations, with IC50 values of 1.15 µM and 19.20 µM, respectively. Furthermore, these compounds significantly inhibited SARS-CoV-2 infection in Vero cells, presenting IC50 values of 11.31 µM for (+)-miliusol and 17.92 µM for miliusane XXXVI. Additionally, both compounds exhibited a potent antiviral effect against the emergent mosquito-borne Zika virus, with IC50 values of 1.34 µM and 23.45 µM, respectively. Time-of-addition assays suggest that their mechanism of action might target later stages of the viral cycle, indicating potential modulation of specific cellular pathways. These findings reinforce the invaluable contribution of medicinal flora as reservoirs of natural antiviral agents and emphasize their prospective role in combatting viruses of medical interest.

(-)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry.

UNLABELLED: Here, we identify (-)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 µM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. CONCLUSION: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte.