Hepatitis C Virus NS3-4A Protease Research Articles

Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

Telaprevir-based Triple Therapy for Retreatment of Chronic Hepatitis C Patients with Genotype Four Followed in Our Clinic

OZ Objective: Telaprevir-based triple therapy for chronic hepatitis C virus (HCV) patients with genotype 4 is not recommended potently by the guidelines. There are few studies in the literature related to this issue. This study showed the antiviral activity and safety of telaprevir-based regimens in the treatment of treatmentexperienced genotype 4 chronic HCV-infected patients. Materials and Methods: This retrospective study consisted of 12 genotype 4 HCV-infected patients. All patients received 12 weeks of telaprevir in combination with 24 weeks of pegylated interferon (PEG-IFN) alpha and ribavirin (RBV). Results: The sustained virological response (SVR) rate was six of 12 (50%). Notably, the rate of SVR in prior relapsers was 75% (6 of 8). SVR could not be achieved in non-responders. Conclusion: Telaprevir, a potent HCV NS3-4A protease inhibitor, has been used as monotherapy and in combination with PEG-IFN/RBV in patients infected with genotype 1, 2 and 3 HCV. Limited clinical data suggest that telaprevir has activity against genotype 4 HCV. In this study, it was observed that the addition of telaprevir to the standard regimen had a greater activity on treatment relapse patients with genotype 4.

Identification of Peptide Leads to Inhibit Hepatitis C Virus: Inhibitory Effect of Plectasin Peptide Against Hepatitis C Serine Protease

The emerging of hepatitis C virus (HCV) resistant strains has been considered as a main drawback of the available drugs. Since HCV has a large inactive surface, we would like to hypothesis that the mutation occur in HCV is minimal and causing less resistance against inhibition. In this study, a short peptide inhibitor of HCV namely plectasin was identified by HCV NS3-4A serine protease assay. Plectasin peptide showed considerable inhibition against HCV NS3-4A serine protease. Enzymatic activity of the recombinant NS3-4Apro was analysed by fluorescence release from several fluorogenic peptide substrates which resembling the dibasic cleavage site sequences of the flavivirus polyprotein precursor. Of all amc-labelled peptides, Pyr-RTKR-amc was the most efficiently cleaved substrate with the lowest Km value of 20 µM. The kinetic assay showed that plectasin peptide inhibited NS3-4Apro activity with an IC50 value of 4.3 μM compared to the aprotinin as a standard proteases inhibitor with an IC50 of 6.1 μM. From the results, plectasin peptide also demonstrated a dose-dependent inhibition of HCV replication with a considerable reduction in RLuc activity at 15 µM using HCV replicon- containing Huh-7 cells. Our study has identified a unique natural peptide that can be used to highlight novel structures for the development of drug derivatives with high efficacy of HCV NS3-4A protease inhibitors.

Control of hepatitis C virus replication in mouse liver-derived cells by MAVS-dependent production of type I and type III interferons.

Hepatitis C virus (HCV) efficiently infects only humans and chimpanzees. Although the detailed mechanisms responsible for this narrow species tropism remain elusive, recent evidence has shown that murine innate immune responses efficiently suppress HCV replication. Therefore, poor adaptation of HCV to evade and/or counteract innate immune responses may prevent HCV replication in mice. The HCV NS3-4A protease cleaves human MAVS, a key cellular adaptor protein required for RIG-I-like receptor (RLR)-dependent innate immune signaling. However, it is unclear if HCV interferes with mouse MAVS function equally well. Moreover, MAVS-dependent signaling events that restrict HCV replication in mouse cells were incompletely defined. Thus, we quantified the ability of HCV NS3-4A to counteract mouse and human MAVS. HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Moreover, replicon-encoded protease cleaved a similar fraction of both MAVS variants. Finally, FLAG-tagged MAVS proteins repressed HCV replication to similar degrees. Depending on MAVS expression, HCV replication in mouse liver cells triggered not only type I but also type III IFNs, which cooperatively repressed HCV replication. Mouse liver cells lacking both type I and III IFN receptors were refractory to MAVS-dependent antiviral effects, indicating that the HCV-induced MAVS-dependent antiviral state depends on both type I and III IFN receptor signaling. In this study, we found that HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Therefore, it is unlikely that ineffective cleavage of mouse MAVS per se precludes HCV propagation in immunocompetent mouse liver cells. Hence, approaches to reinforce HCV replication in mouse liver cells (e.g., by expression of essential human replication cofactors) should not be thwarted by the poor ability of HCV to counteract MAVS-dependent antiviral signaling. In addition, we show that mouse MAVS induces both type I and type III IFNs, which together control HCV replication. Characterization of type I or type III-dependent interferon-stimulated genes in these cells should help to identify key murine restriction factors that preclude HCV propagation in immunocompetent mouse liver cells.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease.

Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus

Infection with hepatitis C virus (HCV) is a major medical problem with over 170 million people infected worldwide. Substantial morbidity and mortality are associated with hepatic manifestations (cirrhosis and hepatocellular carcinoma), which develop with increasing frequency in people infected with HCV for more than 20 years. Less well known is the burden of HCV disease associated with extrahepatic manifestations (diabetes, B-cell proliferative disorders, depression, cognitive disorders, arthritis and Sjögren's syndrome). For patients infected with genotype 1 HCV, treatment with polyethylene glycol decorated interferon (peginterferon) α and ribavirin (PR) is associated with a low (40-50%) success rate, substantial treatment-limiting side effects and a long (48-week) duration of treatment. In the past 15 years, major scientific advances have enabled the development of new classes of HCV therapy, the direct-acting antiviral agents, also known as specifically targeted antiviral therapy for hepatitis C (STAT-C). In combination with PR, the HCV NS3-4A protease inhibitor telaprevir has recently been approved for treatment of genotype 1 chronic HCV in the United States, Canada, European Union and Japan. Compared with PR, telaprevir combination therapy offers significantly improved viral cure rates and the possibility of shortened treatment duration for diverse patient populations. Developers of innovative drugs have to blaze a new path with few validated sign posts to guide the way. Indeed, telaprevir's development was once put on hold because of its performance in a standard IC(50) assay. Data from new hypotheses and novel experiments were required to justify further investment and reduce risk that the drug might fail in the clinic. In addition, the poor drug-like properties of telaprevir were a formidable hurdle, which the manufacturing and formulation teams had to overcome to make the drug. Finally, novel clinical trial designs were developed to improve efficacy and shorten treatment in parallel instead of sequentially. Lessons learned from the development of telaprevir suggest that makers of innovative medicines cannot rely solely on traditional drug discovery metrics, but must develop innovative, scientifically guided pathways for success.

A new method for induced fit docking (genius) and its application to virtual screening of novel HCV NS3-4A protease inhibitors

Hepatitis C virus (HCV) is an etiologic agent of chronic liver disease, and approximately 170 million people worldwide are infected with the virus. HCV NS3-4A serine protease is essential for the replication of this virus, and thus has been investigated as an attractive target for anti-HCV drugs. In this study, we developed our new induced-fit docking program (genius), and applied it to the discovery of a new class of NS3-4A protease inhibitors (IC50=1–10μM including high selectivity index). The new inhibitors thus identified were modified, based on the docking models, and revealed preliminary structure–activity relationships. Moreover, the genius in silico screening performance was validated by using an enrichment factor. We believe our designed scaffold could contribute to the improvement of HCV chemotherapy.

Establishment and application of high throughput screening model for hepatitis C virus NS3-4A protease inhibitors in vitro.

To establish fluorescence resonance energy transfer (FRET) assay method of detecting proteolytic activity of non-structural protein 3-4A (NS3-4A) serine protease of hepatitis C virus (HCV) for high throughput screening inhibitors against HCV in vitro. HCV recombinant plasmid pMAL~c2/NS3-4A was transformed into the E.coli strain K12TB1. Maltose-binding-protein (MBP) NS3-4A fusion protein expression was induced by adding isopropyl-β-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The proteolytic activity of MBP-NS3-4A protease was analyzed by FRET with the special protease substrate. The reaction system in this model was optimized, and the reliability of the model was evaluated. High throughput screening model for HCV NS3-4A protease inhibitors was established, and the best concentrations of enzyme and substrate were optimized. In the model, the Km value of protease was 4.74 μmol/L, Z factor was up to 0.80, and coefficient of variation (CV) was 1.91%. BILN 2061, one of the known HCV protease inhibitors, was measured with the Ki of 0.30 nmol/L. The assay model using FRET method for HCV NS3 4A serine protease is stable and reliable, and the model is suitable for high throughput screening for HCV NS3 4A protease inhibitors.

Elimination of hepatitis C virus by short term NS3-4A and NS5B inhibitor combination therapy in human hepatocyte chimeric mice

The current treatment regimen for chronic hepatitis C virus (HCV) infection is peg-interferon plus ribavirin combination therapy. The majority of developing therapeutic strategies also contain peg-interferon with or without ribavirin. However, interferon is expensive and sometimes intolerable for some patients because of severe side effects. Using human hepatocyte chimeric mice, we examined whether a short term combination therapy with the HCV NS3-4A protease inhibitor telaprevir and the RNA polymerase inhibitor MK-0608 with or without interferon eradicates the HCV from infected mice. The effect of telaprevir and MK-0608 combination therapy was examined using subgenomic HCV replicon cells. Combination therapy with the two drugs enhanced inhibition of HCV replication compared with either drug alone. In in vivo experiments, early emergence of drug resistance was seen in mice treated with either telaprevir or MK-0608 alone. However, emergence was prevented by the combination of these drugs. Mice treated with a triple combination therapy of telaprevir, MK-0608, and interferon became negative for HCV RNA soon after commencement of the therapy, and HCV RNA was not detected in serum of these mice 12 weeks after cessation of the therapy. Furthermore, all mice treated with a high dose telaprevir and MK-0608 combination therapy for 4 weeks became negative for HCV RNA 1 week after the beginning of the therapy and remained negative after 18 weeks. Eradication of HCV from mice with only 4 weeks of therapy without interferon points the way to future combination therapies for chronic hepatitis C patients.

Binary selectivity for HCV NS3/4A protease versus host proteases via irreversible inactivation at a non‐catalytic cysteine

Viral protease inhibitors typically achieve potency via interactions with the catalytic apparatus, but as a result are often poorly selective. Viral protease inhibitors must also block emerging mutants. We have developed a fundamentally different approach to the design of covalent inhibitors by targeting non‐catalytic cysteine residues unique to viral targets but absent in host proteases, enabling extreme, “binary” selectivity. An estimated 170 million people worldwide are infected with hepatitis C virus (HCV). HCV NS3/4A protease (HCVP) is a key target for HCV therapy. We have identified a class of irreversible HCVP inhibitors that bond covalently to the non‐catalytic Cys159 in HCVP. Cys159 is conserved across all HCV genotypes but is absent in host proteases. MALDI‐TOF MS and in vitro assays show that the lead inhibitor bonds to and inhibits HCVP across multiple genotypes and drug‐resistant variants. In replicon assays covalent silencing leads to potent inhibition of HCVP and prolonged duration of action (>24 hrs) after brief exposure to inhibitor. Covalent silencing yields potent inhibition of HCVP in vivo, in a new mouse model. Inhibitors with this profile may profoundly improve hepatitis C patient treatment across all HCV genotypes. This design strategy is also readily applicable to a wide range of therapeutically important proteases. This work was supported by Avila Therapeutics.

Cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis C correlates with a reduced activation of the endogenous interferon system

Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-alpha. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta. The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS.

Mutation rate in hepatitis C virus NS3 protease is not influenced by HIV-1 protease inhibitor therapy

Mutation rate in hepatitis C virus NS3 protease is not influenced by HIV-1 protease inhibitor therapy

VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log(10) was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log(10) reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

Preclinical Profile of VX-950, a Potent, Selective, and Orally Bioavailable Inhibitor of Hepatitis C Virus NS3-4A Serine Protease

VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.

Discovery of Small-Molecule Inhibitors of HCV NS3-4A Protease as Potential Therapeutic Agents against HCV Infection

Chronic infection with hepatitis C virus (HCV) is associated with liver cirrhosis that often leads to hepatic failure and hepatocellular carcinoma (HCC). HCV infection has become a global health threat and the main cause of adult liver transplants in developed nations. Current approved anti-HCV therapies (interferon and pegylated interferon alone or in combination with ribavirin) are not effective in eliminating the viral infection in a significant population of patients (e.g., those infected with HCV genotype 1). Furthermore, these therapies are plagued with many undesirable side effects. Therefore, the HCV epidemic represents a huge unmet medical need that has triggered intensive research efforts towards the development of more effective drugs. Given its essential role in the process of HCV replication, the viral NS3/4A serine protease is arguably the most thoroughly characterized HCV enzyme and the most intensively pursued anti-HCV target for drug development. This is further fueled by the successful use of small-molecule inhibitors of the human immunodeficiency virus (HIV) viral protease, which have had an impressive effect on HIV-related morbidity and mortality, offering hope that analogous drugs might also have a similar impact against HCV. Here, we review the recent progress and development of small-molecule inhibitors of the HCV NS3/4A protease. In particular, we focus on the discovery of VX-950, the latest HCV NS3-4A protease inhibitor to be advanced to clinical studies. While the challenges of designing potent inhibitors of the viral protease have been solved, as highlighted by BILN 2061 and VX-950, it is still too early to determine whether these efforts will eventually yield promising drug candidates. For the emerging small-molecule HCV inhibitors, viral resistance will likely be a big problem. Thus, combination therapy of different drugs with different targets/mechanisms will be necessary to effectively inhibit HCV replication. It is also hoped that a detail characterization of how the resistance mutations that affect NS3 inhibitor binding may provide useful information for the design of inhibitors with the potential to treat resistant viruses that may arise during chronic HCV infection.