anti-HCV Molecules Research Articles

Synthesis, Characterization and Docking Studies of Anti-HCV Molecules

The present study reports the synthesis and characterization of novel molecules inhibiting the spread of hepatitis C. The molecules were designed as to block the NS3/4A protease enzyme on HCV RNA. The molecules were synthesized using usual peptide synthesis techniques. Compounds with purity more than 95% were characterized and docking studies were also performed. All the compounds were characterized using physico-chemical techniques such as determination of melting point by DSC and NMR, mass, IR spectral studies. The docking studies were also conducted to assess the activity of molecules for inhibition of hepatitis C virus.

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

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. 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.

The discovery and characterization of a novel scaffold as a potent hepatitis C virus inhibitor.

HCV infections are a major global health concern. Although direct acting antiviral agents have significantly improved the response rate of anti-HCV therapy, they also suffer from drug resistance, unfavorable pharmacokinetic profiles and high costs. Thus, it is still highly desirable to develop new anti-HCV therapeutics. Herein a novel anti-HCV benzothiazole scaffold was discovered by phenotypic screening. Further target characterization and structural optimization studies revealed that the benzothiazole-disulfoamide derivatives were potent anti-HCV molecules with good selectivity and acted by targeting NS5A.

Design, synthesis and evaluation of novel anti-HCV molecules that deliver intracellularly three highly potent NS5A inhibitors.

The design and synthesis of new non-symmetrical NS5A inhibitors with sulfur containing amino acids is reported along with their ability to block HCV replication in an HCV 1b replicon system. These compounds display EC50 values in the picomolar range with a large therapeutic index (>10(6)). Moreover, cellular pharmacology studies show that our preferred compounds intracellularly deliver three potent NS5A inhibitors.

The antimalarial ferroquine is an inhibitor of hepatitis C virus

Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti‐HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)‐free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell‐cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell‐free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell‐to‐cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. Conclusion: FQ is a novel, interesting anti‐HCV molecule that could be used in combination with other direct‐acting antivirals. (Hepatology 2013)

VAPC, an Human Endogenous Inhibitor for Hepatitis C Virus (HCV) Infection, Is Intrinsically Unstructured but Forms a “Fuzzy Complex” with HCV NS5B

Nearly 200 million people are infected by hepatitis C virus (HCV) worldwide. For replicating the HCV genome, the membrane-associated machinery needs to be formed by both HCV non-structural proteins (including NS5B) and human host factors such as VAPB. Recently, the 99-residue VAPC, a splicing variant of VAPB, was demonstrated to inhibit HCV replication via binding to NS5B, thus acting as an endogenous inhibitor of HCV infection. So far, the structure of VAPC remains unknown, and its interaction with NS5B has not been biophysically characterized. In this study, we conducted extensive CD and NMR investigations on VAPC which led to several striking findings: 1) although the N-terminal 70 residues are identical in VAPC and VAPB, they constitute the characteristic β-barrel MSP fold in VAPB, while VAPC is entirely unstructured in solution, only with helical-like conformations weakly populated. 2) VAPC is indeed capable of binding to NS5B, with an average dissociation constant (Kd) of ∼20 µM. Intriguingly, VAPC remains dynamic even in the complex, suggesting that the VAPC-NS5B is a “fuzzy complex”. 3) NMR mapping revealed that the major binding region for NS5B is located over the C-terminal half of VAPC, which is composed of three discrete clusters, of which only the first contains the region identical in VAPC and VAPB. The second region containing ∼12 residues appears to play a key role in binding since mutation of 4 residues within this region leads to almost complete loss of the binding activity. 4) A 14-residue mimetic, VAPC-14 containing the second region, only has a ∼3-fold reduction of the affinity. Our study not only provides critical insights into how a human factor mediates the formation of the HCV replication machinery, but also leads to design of VAPC-14 which may be further used to explore the function of VAPC and to develop anti-HCV molecules.

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

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. 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.

Management of nonresponsive hepatitis C

More than 50% of hepatitis C virus (HCV)-infected patients do not respond to the classical pegylated interferon (PEG-IFN)/ribavirin combination therapy. However, failing to respond to one course of treatment is not synonymous of therapy failure and retreatment is often beneficial. Alternative retreatment strategies include repeating the classical standard of care with an optimized drug regimen and adherence, including ribavirin serum concentration adjustment, correcting, if at all possible, comorbidities, and the addition of new specific anti-HCV molecules to the backbone of pegylated interferon/ribavirin. Options of retreatment should include consensus and natural interferons. For patients with advanced disease exposed to a high risk of lethal complications, customized maintenance therapy could be an effective option since it may slow down complications in some patients. Since low-dose interferon monotherapy is not sufficient, such a maintenance therapy remains to be verified via clinical trials. New possibilities of noninvasive assessment of fibrosis and the use of genetic tests to predict fibrosis progression and responsiveness to interferon are major emerging opportunities that run parallel to the revolution of the pharmacologic armentarium.

Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20-fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2-fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors.

Antiviral therapy: chronic hepatitis C

The combination of pegylated interferon (IFN) with ribavirin is the standard of care for chronic hepatitis C. Response rates range from 50 to 90% for genotype 1 and 2/3 which also differ in the duration of treatment (48 vs 24 weeks, respectively). Therapy if given with weight-based dosing may be shortened from 24 to 12, 14 or 16 weeks (genotypes 2 and 3), and from 48 to 24 weeks (genotype 1) in case of hepatitis C virus (HCV) clearance at week 4, without reducing sustained virologic response (SVR). Conversely, prolonging treatment for 72 weeks in those with only a decrease of viral load at week 4, i.e. "slow responders" increases SVR rates by preventing relapse. In spite of such progress, over half of patients are relapsers or nonresponders. If the previous treatment was suboptimal, retreatment with higher doses or longer duration may be beneficial unless an individual was a 'null responder' previously. New promising anti-HCV molecules (antiproteases and antipolymerases), some with potent antiviral activity, are in phase II trials but appear to require the addition of IFN and ribavirin to maintain viral suppression. Other immunomodulatory agents such as new IFN or therapeutic vaccines and alternatives to ribavirin are also under development. Future regimens should improve efficacy and provide shorter and better-tolerated combination therapy.