Hepatitis C Virus Entry Inhibitors Research Articles

Design, Synthesis, and Biological Evaluation of 2-((4-Bisarylmethyl-piperazin-1-yl)methyl)benzonitrile Derivatives as HCV Entry Inhibitors.

Viral entry inhibitors are absent in hepatitis C virus (HCV) treatment regimens although a dozen direct-acting antiviral (DAA) drugs are available now. Based on a previously identified HCV entry inhibitor L0909, chemical space exploration and structure-activity relationship (SAR) studies led to the discovery of a new derived scaffold 2-((4-bisarylmethyl-piperazin-1-yl)methyl)benzonitrile. Several new scaffold derivatives exhibited higher in vitro anti-HCV activity at low nanomolar concentrations compared to L0909. A biological study indicated that the high potency of active derivatives 3d, 3h, and 3i was primarily driven by the inhibitory effect on the virus entry stage. Moreover, an SPR experiment confirmed that this class of derivatives might target the HCV E1 protein. Pharmacokinetic studies indicated that compounds 3d and 3i are orally available and long-lasting in rat plasma after oral administration to rats by a single dose of 15 mg/kg. In conclusion, this work provided a novel 2-((4-bisarylmethyl-piperazin-1-yl)methyl)benzonitrile chemotype deserving further investigation into its antiviral therapeutic potential.

Validated High-performance Liquid Chromatography Method for the Determination of JW5624, A New Class Of Hepatitis C Virus Inhibitor, in Rat Plasma and its Application in the Pharmacokinetic Study of JW5624.

We developed unique small-molecule inhibitors of hepatitis C virus (HCV), which had potent activity for HCV entry inhibition and multi-genotypic antiviral activity. In this study, a sensitive and reliable method for the quantitation of JW5624 in rat plasma was developed and validated using high performance liquid chromatography. Chromatographic separation was achieved using a reversed-phase (C18) column. The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (30:70, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil (an internal standard), and JW5624 were approximately 5.9 and 7.3 min, respectively. The detection limit of JW5624 in rat plasma was 0.03 μg/mL. Pharmacokinetic parameters of JW5624 was evaluated after intravenous (i. v.; at doses of 5 mg/kg) and oral (p.o.; at doses of 10 mg/kg) administration of JW5624 in rats. After p.o. administration (10 mg/kg) of JW5624, F value was approximately 71.0%. These results suggest that JW5624 can be a potential candidate drug for the development of HCV entry inhibitors.

2-((4-Arylpiperazin-1-yl)methyl)benzonitrile Derivatives as Orally Available Inhibitors of Hepatitis C Virus with a Novel Mechanism of Action.

Although the direct-acting antivirals revolutionized the hepatitis C virus (HCV) infection treatment in the last decade, more efforts are needed to reach the elimination of HCV in the absence of a vaccine. 4-(Piperazin-1-yl)-2-((p-tolylamino)methyl)-benzonitrile (1) is a modest HCV inhibitor identified from an in-house screening using a HCV-infected Huh7.5 cell culture. Starting from it, the chemical optimization afforded a new 2-((4-arylpiperazin-1-yl)methyl)benzonitrile scaffold with significantly increased antiviral activity against HCV. A highly effective HCV inhibitor, 35 (L0909, EC50 = 0.022 μM, SI > 600), was identified by the structure-activity relationship study. The biological study revealed that L0909 could block HCV replication by acting on the HCV entry stage. The high sensitivity to clinical resistant HCV mutants and synergistic effect with clinical drugs were observed for this compound. The further pharmaceutical studies demonstrated that L0909 is long-lasting, is orally available, and has low toxicity in vivo. These results show L0909 as a promising HCV entry inhibitor for single or combinational therapeutic potential.

Short-course, direct-acting antivirals and ezetimibe to prevent HCV infection in recipients of organs from HCV-infected donors: a phase 3, single-centre, open-label study

An increasing percentage of potential organ donors are infected with hepatitis C virus (HCV). After transplantation from an infected donor, establishment of HCV infection in uninfected recipients is near-universal, with the requirement for post-transplant antiviral treatment. The aim of this study was to determine if antiviral drugs combined with an HCV entry blocker given before and for 7 days after transplant would be safe and reduce the likelihood of HCV infection in recipients of organs from HCV-infected donors. HCV-uninfected organ recipients without pre-existing liver disease were treated with ezetimibe (10 mg; an HCV entry inhibitor) and glecaprevir-pibrentasvir (300 mg/120 mg) before and after transplantation from HCV-infected donors aged younger than 70 years without co-infection with HIV, hepatitis B virus, or human T-cell leukaemia virus 1 or 2. Recipients received a single dose 6-12 h before transplant and once a day for 7 days after surgery (eight doses in total). HCV RNA was assessed once a day for 14 days and then once a week until 12 weeks post-transplant. The primary endpoint was prevention of chronic HCV infection, as evidenced by undetectable serum HCV RNA at 12 weeks after transplant, and assessed in the intention-to-treat population. Safety monitoring was according to routine post-transplant practice. 12-week data are reported for the first 30 patients. The trial is registered on ClinicalTrials.gov, NCT04017338. The trial is closed to recruitment but follow-up is ongoing. 30 patients (23 men and seven women; median age 61 years (IQR 48-66) received transplants (13 lung, ten kidney, six heart, and one kidney-pancreas) from 18 HCV-infected donors. The median donor viral load was 5·11 log10IU/mL (IQR 4·55-5·63) and at least three HCV genotypes were represented (nine [50%] donors with genotype 1, two [11%] with genotype 2, five [28%] with genotype 3, and two [11%] with unknown genotype). All 30 (100%) transplant recipients met the primary endpoint of undetectable HCV RNA at 12 weeks post-transplant, and were HCV RNA-negative at last follow-up (median 36 weeks post-transplant [IQR 25-47]). Low-level viraemia was transiently detectable in 21 (67%) of 30 recipients in the early post-transplant period but not after day 14. Treatment was well tolerated with no dose reductions or treatment discontinuations; 32 serious adverse events occurred in 20 (67%) recipients, with one grade 3 elevation in alanine aminotransferase (ALT) possibly related to treatment. Non-serious transient elevations in ALT and creatine kinase during the study dosing period resolved with treatment completion. Among the serious adverse events were two recipient deaths due to causes unrelated to study drug treatment (sepsis at 49 days and subarachnoid haemorrhage at 109 days post-transplant), with neither patient ever being viraemic for HCV. Ezetimibe combined with glecaprevir-pibrentasvir given one dose before and for 7 days after transplant prevented the establishment of chronic HCV infection in recipients of different organs from HCV-infected donors. This study shows that an ultra-short course of direct-acting antivirals and ezetimibe can prevent the establishment of chronic HCV infection in the recipient, alleviating many of the concerns with transplanting organs from HCV-infected donors. Canadian Institutes of Health Research; the Organ Transplant Program, University Health Network.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

Inhibition Mechanisms of Hepatitis C Virus Infection by Caffeic Acid and Tannic Acid.

Previously, we reported that coffee extract and its constituents, caffeic acid (CA) and p-coumaric acid, inhibit infection by the hepatitis C virus (HCV). In the present report, we identified another coffee-related compound, tannic acid (TA), which also inhibits HCV infection. We systematically evaluated which steps of the viral lifecycle were affected by CA and TA. TA substantially inhibits HCV RNA replication and egression, while CA does not. The infectivity of the HCV pretreated with CA or TA was almost lost. Cellular attachment of HCV particles and their interaction with apolipoprotein E, which is essential for HCV infectivity, were significantly reduced by CA. These results indicate that CA inhibits HCV entry via its direct effect on viral particles and TA inhibits HCV RNA replication and particle egression as well as entry into host cells. Taken together, our findings may provide insights into CA and TA as potential anti-HCV strategies.

Farnesoid X receptor agonist GW4064 indirectly inhibits HCV entry into cells via down-regulating scavenger receptor class B type I

Farnesoid X receptor (FXR) agonists play important regulatory roles in bile acid, lipid and glucose metabolism in vitro and in vivo. Thus, FXR agonists exhibit potential therapeutic effects on metabolism-related diseases that are associated with extrahepatic manifestations induced by hepatitis C virus (HCV) infection. This study investigated the effect and mechanism of FXR agonist GW4064 against HCV in vitro to explore the potential application of FXR agonists. Results showed that GW4064 and other FXR agonists have potent antiviral activity against HCV in Huh7.5 cells. GW4064 down-regulated the expression of scavenger receptor class B type I protein via FXR and thereby indirectly inhibited HCV entry into cells, leading to interruption of HCV life cycle. GW4064 also exhibited synergistic anti-HCV effect with known direct-acting antiviral agents (DAAs) used in the clinic and remained sensitive to DAA-resistant HCV mutations. Therefore, FXR agonists are also a kind of antiviral agent, and might be helpful in treatment of HCV-induced hepatic and extrahepatic manifestations.

A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors

Hepatitis C virus (HCV) infection causes chronic liver disease. Antivirals have been developed and cure infection. However, resistance can emerge and salvage therapies with alternative modes of action could be useful. Several licensed drugs have emerged as HCV entry inhibitors and are thus candidates for drug repurposing. We aimed to dissect their mode of action, identify improved derivatives and determine their viral targets. HCV entry inhibition was tested for a panel of structurally related compounds, using chimeric viruses representing diverse genotypes, in addition to viruses containing previously determined resistance mutations. Chemical modeling and synthesis identified improved derivatives, while generation of susceptible and non-susceptible chimeric viruses pinpointed E1 determinants of compound sensitivity. Molecules of the diphenylpiperazine, diphenylpiperidine, phenothiazine, thioxanthene, and cycloheptenepiperidine chemotypes inhibit HCV infection by interfering with membrane fusion. These molecules and a novel p-methoxy-flunarizine derivative with improved efficacy preferentially inhibit genotype 2 viral strains. Viral residues within a central hydrophobic region of E1 (residues 290-312) control susceptibility. At the same time, viral features in this region also govern pH-dependence of viral membrane fusion. Small molecules from different chemotypes related to flunarizine preferentially inhibit HCV genotype 2 membrane fusion. A hydrophobic region proximal to the putative fusion loop controls sensitivity to these drugs and the pH range of membrane fusion. An algorithm considering viral features in this region predicts viral sensitivity to membrane fusion inhibitors. Resistance to flunarizine correlates with more relaxed pH requirements for fusion. This study describes diverse compounds that act as HCV membrane fusion inhibitors. It defines viral properties that determine sensitivity to these molecules and thus provides information to identify patients that may benefit from treatment with membrane fusion inhibitors.

Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle.

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle.

Pentagalloylglucose, a highly bioavailable polyphenolic compound present in Cortex moutan, efficiently blocks hepatitis C virus entry

Approximately 142 million people worldwide are infected with hepatitis C virus (HCV). Although potent direct acting antivirals are available, high costs limit access to treatment. Chronic hepatitis C virus infection remains a major cause of orthotopic liver transplantation. Moreover, re-infection of the graft occurs regularly. Antivirals derived from natural sources might be an alternative and cost-effective option to complement therapy regimens for global control of hepatitis C virus infection.We tested the antiviral properties of a mixture of different Chinese herbs/roots named Zhi Bai Di Huang Wan (ZBDHW) and its individual components on HCV. One of the ZBDHW components, Penta-O-Galloyl-Glucose (PGG), was further analyzed for its mode of action in vitro, its antiviral activity in primary human hepatocytes as well as for its bioavailability and hepatotoxicity in mice.ZBDHW, its component Cortex Moutan and the compound PGG efficiently block entry of HCV of all major genotypes and also of the related flavivirus Zika virus. PGG does not disrupt HCV virion integrity and acts primarily during virus attachment. PGG shows an additive effect when combined with the well characterized HCV inhibitor Daclatasvir. Analysis of bioavailability in mice revealed plasma levels above tissue culture IC50 after a single intraperitoneal injection.In conclusion, PGG is a pangenotypic HCV entry inhibitor with high bioavailability. The low cost and wide availability of this compound make it a promising candidate for HCV combination therapies, and also emerging human pathogenic flaviviruses like ZIKV.

Sec24C-Dependent Transport of Claudin-1 Regulates Hepatitis C Virus Entry.

Claudin-1 is a hepatitis C virus (HCV) coreceptor required for viral entry. Although extensive studies have focused on claudin-1 as an anti-HCV target, little is known about how the level of claudin-1 at the cell surface is regulated by host vesicular transport. Here, we identified an interaction between claudin-1 and Sec24C, a cargo-sorting component of the coat protein complex II (COPII) vesicular transport system. By interacting with Sec24C through its C-terminal YV, claudin-1 is transported from the endoplasmic reticulum (ER) and is eventually targeted to the cell surface. Blocking COPII transport inhibits HCV entry by reducing the level of claudin-1 at the cell surface. These findings provide mechanistic insight into the role of COPII vesicular transport in HCV entry.IMPORTANCE Tight junction protein claudin-1 is one of the cellular receptors for hepatitis C virus, which infects 185 million people globally. Its cellular distribution plays important role in HCV entry; however, it is unclear how the localization of claudin-1 to the cell surface is controlled by host transport pathways. In this paper, we not only identified Sec24C as a key host factor for HCV entry but also uncovered a novel mechanism by which the COPII machinery transports claudin-1 to the cell surface. This mechanism might be extended to other claudins that contain a C-terminal YV or V motif.

A cinnamon-derived procyanidin type A compound inhibits hepatitis C virus cell entry.

Chronic hepatitisC virus (HCV) infection is a major cause of liver disease worldwide. Although direct-acting antivirals can cure the large majority of treated patients, important limitations remain, including treatment failure and high costs precluding access to therapy in resource-limited settings. We report herein the anti-HCV effects of IND02, a procyanidin typeA molecule, isolated and characterized from cinnamon. Using cellculture-derived HCV (HCVcc), HCV pseudoparticles (HCVpp), and subgenomic replicons, we demonstrated that IND02 markedly and dose-dependently inhibited HCV cell entry. Kinetic assays demonstrated that IND02 inhibits HCV entry most likely at a postbinding step. Experiments performed using primary human hepatocytes confirmed inhibition of HCV entry by IND02, demonstrating the functional impact in the most physiological cell-based system for studying HCV-host interactions. The natural compound IND02 exhibits potent HCV cell entry inhibition and provides a novel perspective for development of a low-cost antiviral for treatment of HCV infection.

Identification of a flavonoid isolated from plum (Prunus domestica) as a potent inhibitor of Hepatitis C virus entry

Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases that often requires liver transplantation. The standard therapies are limited by severe side effects, resistance development, high expense and in a substantial proportion of cases, fail to clear the infection which bespeak the need for development of well-tolerated antivirals. Since most of the drug development strategies target the replication stage of viral lifecycle, the identification of entry inhibitors might be crucial especially in case of liver-transplant recipients. In the present study we have evaluated fruits which are known for their hepatoprotective effects in order to screen for entry inhibitors. We report the identification of a flavonoid, rutin, isolated from Prunus domestica as a new HCV entry inhibitor. Characterization and confirmation of the chemical structure was done by LC-ESI-MS, NMR and IR spectral analyses. Rutin significantly inhibited HCV-LP binding to hepatoma cells and inhibited cell-culture derived HCV (HCVcc) entry into hepatoma cells. Importantly, rutin was found to be non-toxic to hepatoma cells. Furthermore, rutin inhibits the early entry stage of HCV lifecycle possibly by directly acting on the viral particle. In conclusion, rutin is a promising candidate for development of anti-HCV therapeutics in the management of HCV infection.

A screen for inhibitory peptides of hepatitis C virus identifies a novel entry inhibitor targeting E1 and E2

Hepatitis C virus (HCV) entry into hepatocytes is a multistep process that represents a promising target for antiviral intervention. The viral envelope protein E1E2 plays a critical role in HCV entry. In this study, we sought to identify peptide inhibitors of HCV by screening a library of overlapping peptides covering E1E2. Screening the peptide library identified several novel anti-HCV peptides. Four peptides from glycoprotein E2 were selected for further investigation. The 50% effective dose (ED50) was approximately 5 nM for each peptide. Our data indicated that these peptides inhibited HCV entry at the post-attachment step. Moreover, these peptides blocked cell-to-cell transmission of HCVcc and had broad-spectrum antiviral effects on HCVcc. These peptides exhibited combination inhibitory effects on HCVcc infection when combined with IFN-α2b or anti-CD81 antibody. Interestingly, we observed that E2-42 associated with E1 and E2. Our results indicate that E2-42 inhibits HCV entry via E1 and E2. These findings suggest a new avenue for HCV therapeutic development.

The role of PTEN - HCV core interaction in hepatitis C virus replication

Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid phosphatase activity. PTEN has no effect on HCV RNA translation. PTEN decreases HCV replication and the protein phosphatase activity of PTEN is essential for this function. PTEN interacts with the HCV core protein and requires R50 in domain I of HCV core and PTEN residues 1–185 for this interaction. This interaction is required for PTEN-mediated inhibition of HCV replication. This gives rise to a reduction in PTEN levels and intracellular lipid abundance, which may in turn regulate HCV replication. HCV core domain I protein increases the lipid phosphatase activity of PTEN in an in vitro assay, suggesting that HCV infection can also regulate PTEN. Taken together, our results demonstrated an important regulatory role of PTEN in the HCV life cycle.

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug‐resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size‐exclusion chromatography, we obtained from HCV‐seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low‐density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma‐derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine‐binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851‐1864)

A plant-derived dehydrorotenoid: a new inhibitor of hepatitis C virus entry.

Emergence of drug-resistant viruses, high cost and adverse side-effects associated with the standard therapy against hepatitis C virus (HCV) infection demonstrate the need for development of well tolerated and effective antivirals. We identified and chemically characterised the dehydrorotenoid boeravinone H, isolated from the herb Boerhaviadiffusa, as a new inhibitor of HCV entry. The compound significantly inhibits the binding and entry of hepatitis C-like particles (HCV-LPs) in hepatoma cells invitro with no apparent cytotoxicity. Boeravinone H inhibits the initial phase of HCV entry probably by acting directly on the viral particle. Importantly, the compound prevents HCV entry and infection in cell culture (exvivo). Thus, boeravinone H is a potential antiviral agent for the prevention and control of HCV infection.

Synthesis and biological evaluation of novel pentacyclic triterpene α-cyclodextrin conjugates as HCV entry inhibitors

Hepatitis C virus (HCV) entry is a key target for the treatment of chronic HCV infection. In our continuing efforts to identify novel potential anti-HCV entry inhibitors, a series of water-soluble triazole-bridged α-cyclodextrin-pentacyclic triterpene conjugates were easily synthesized with moderate to good yields. These novel compounds were fully identified and characterized by 1D and 2D NMR spectroscopy and ESI-HRMS. The anti-HCV entry activities were determined based on HCVpp/VSVGpp entry assays. The best results were found for compounds 15 and 18, which displayed the most promising anti-HCV entry activities with average IC50 values of 1.18 μM and 0.25 μM, respectively. In addition, the in vitro cytotoxicity activity of the two compounds against MDCK cells showed no toxicity at 100 μM. Five different binding assays were set up to identify the action mechanism. The results showed that the compounds exert their inhibitory activity at the post-binding step and subsequently prevent virus entry.

(4R,6S)-2-Dihydromenisdaurilide is a Butenolide that Efficiently Inhibits Hepatitis C Virus Entry.

Without a vaccine, hepatitis C virus (HCV) remains a significant threat, putting 170–300 million carriers worldwide at risk of cirrhosis and hepatocellular carcinoma. Although the direct-acting antivirals targeting HCV replication have revolutionized the treatment of hepatitis C, several obstacles persist, including resistance development, potential side-effects, and the prohibitive cost that limits their availability. Furthermore, treatment of HCV re-infection in liver transplantation remains a significant challenge. Developing novel antivirals that target viral entry could help expand the scope of HCV therapeutics and treatment strategies. Herein, we report (4R,6S)-2-dihydromenisdaurilide (DHMD), a natural butenolide, as an efficient inhibitor of HCV entry. Specifically, DHMD potently inhibited HCV infection at non-cytotoxic concentration. Examination on the viral life cycle demonstrated that DHMD selectively targeted the early steps of infection while leaving viral replication/translation and assembly/release unaffected. Furthermore, DHMD did not induce an antiviral interferon response. Mechanistic dissection of HCV entry revealed that DHMD could inactivate cell-free virus, abrogate viral attachment, and inhibit viral entry/fusion, with the most pronounced effect observed against the viral adsorption phase as validated using ELISA and confocal microscopy. Due to its potency, DHMD may be of value for further development as an entry inhibitor against HCV, particularly for application in transplant setting.

A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes.

Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations.