Hepatitis C Virus NS3 Serine Protease Research Articles

Identification of potential bioactive compounds from Aspergillus terreus against HCV NS3 serine protease.

This study was aimed to isolate bioactive compounds from the fermentation products of Aspergillus terreus, which could inhibit NS3 protease of hepatitis C virus (HCV). The bioactive compounds were isolated by reverse-phase silica-gel column chromatography, semi-preparative reverse-phase, and Sephadex LH-20, and then their structures were elucidated through spectroscopic analysis. As a result, two small molecule compounds were isolated. Compound 1 was identified as a new benzaldehyde, (E)-2,4-dihydroxy-6-propenyl-benzaldehyde. Compound 2 was identified as pleurone, which was obtained from microorganisms for the first time. Their inhibitory activities against HCV NS3 protease (IC50) were 32.6 μM and 78.9 μM, respectively. This study provided a new option for the development of anti-HCV drugs.

Novel inhibitors against wild-type and mutated HCV NS3 serine protease: an in silico study.

In 2011, the FDA approved boceprevir as a hepatitis C virus (HCV) NS3 serine protease inhibitor. The sustained virological response rate for treatment with this approved compound is considerably low. Patients have not responded as much as expected to boceprevir therapy. In this in silico study, modified boceprevir compounds are suggested and tested on wild-type HCV NS3 protease and 19 mutated HCV NS3 proteases using molecular docking. Results reveal the superiority of two of the proposed modified compounds to boceprevir. One of which appears to be more potent than boceprevir itself concerning activity against wild-type NS3 and most of the examined mutated NS3 proteases.

Naturally occurring NS3 resistance-associated variants in hepatitis C virus genotype 1: Their relevance for developing countries

Hepatitis C virus (HCV) is a major cause of global morbidity and mortality, with an estimated 130–150 million infected individuals worldwide. HCV is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options in developing countries involve pegylated interferon-α and ribavirin as dual therapy or in combination with one or more direct-acting antiviral agents (DAA). The emergence of resistance-associated variants (RAVs) after treatment reveals the great variability of this virus leading to a great difficulty in developing effective antiviral strategies. Baseline RAVs detected in DAA treatment-naïve HCV-infected patients could be of great importance for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS3 protease inhibitor mutations has been addressed in many countries, there are only a few reports on their prevalence in South America. In this study, we investigated the presence of RAVs in the HCV NS3 serine protease region by analysing a cohort of Uruguayan patients with chronic hepatitis C who had not been treated with any DAAs and compare them with the results found for other South American countries. The results of these studies revealed that naturally occurring mutations conferring resistance to NS3 inhibitors exist in a substantial proportion of Uruguayan treatment-naïve patients infected with HCV genotype 1 enrolled in these studies. The identification of these baseline RAVs could be of great importance for patients’ management and outcome prediction in developing countries.

Activity of compounds from Taxillus sutchuenensis as inhibitors of HCV NS3 serine protease

This study aimed to isolate active compounds from traditional Chinese medicinal Taxillus sutchuenensis to inhibit hepatitis C virus (HCV) NS3 protease activity. Under the guidance of bioassay, 10 compounds were isolated from the EtOAc extract fraction, which were identified as inhibitors of HCV NS3 protease. IC50 values of these compounds were obtained, and a broad degree of anti-HCV activity was observed. The most active compounds were kaempferol-3,7-bisrhamnoside (19.4 μM) and (3S)-3-hydroxy-1,7-bis(4-hydroxy-phenyl)-6E-hepten-5-one (28.7 μM). In conclusion, flavonoids and diarylheptanoids were responsible for the anti-HCV constitution of Taxilli Herba. These inhibitors of HCV NS3 protease might serve as potential candidate of anti-HCV agents.

A review on novel therapies to combat hepatitis C

The major cause of chronic liver disease, cirrhosis, liver carcinoma and liver failure is Hepatitis C virus (HCV). NS3 is one of the attractive targets for therapy development for HCV as the N-terminal domain of the NS3 protein is a serine protein. NS3 protease acts by interfering with cellular mechanisms which are involved in the host immune response to an HCV infection and the inhibition of the protease is an efficient antiviral approach. NS3 protease inhibitors initially developed were: BILN 2061 (Ciluprevir), VX-950 (Telaprevir) and SCH503034. A combination therapy of injected pegylated interferon-α and oral ribavirin is regarded as the current standard therapy for HCV infection. Leucopenia, flu-like symptoms, thrombocytopenia, depression and anemia are some of the adverse effects of ribavirin. The compounds which constitute a benzoxaborole moiety interact with many biological targets and presents appreciable qualities. The acyl sulfinamide and the acyl cyanamide were shown to be potent P1 C-terminal groups in the enzyme assay. In a study, it is indicated that the aminobenzoyl sulfonamide fragment was identified as a novel P1 structural motif. New P2–P4 macrocycle inhibitors of NS3/4A were made up of a P1 C-terminal carboxylic acid have recently been developed. Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed by incorporating 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a new N-acyl-L-hydroxyproline were developed for the treatment of Hepatitis C. α-Amino cyclic boronates was designed and synthesized and incorporated successfully in several acyclic templates at the P1 position as HCV inhibitors. The boronic acid compounds acts as the HCV NS3 serine protease inhibitors. Some of the HCV-protease inhibitors are developed which are based on a 2(1H)-pyrazinone P3 scaffold in combination with either a P2 phenylglycine or a glycine. NS3/4A protease inhibitors constituting of quinazoline derivatives as P2 substituent were synthesized. The tripeptide-based inhibitors of the HCV NS3 protease containing a novel P2-triazole had been synthesized. A protease domain (NS3pro) and RNA helicase domain (NS3hel) makes a multifunctional enzyme: Flaviviridae non-structural 3 proteins (NS3). Crude ethanol extract from rhizomes of the Chinese medicinal herb Rhodiola kirilowii (Regel) Maxim was also used for the cure of Hepatitis C. A new series of HCV NS3/4A protease inhibitors bearing a P2-P4 macrocycle and a P1- P10 a-ketoamide serine trap has been studied. Key words: Hepatitis C virus, non-structural protein inhibitors.

Molecular dynamics study on drug resistance mechanism of HCV NS3/4A protease inhibitor: BI201335

Hepatitis C virus (HCV) infection is a serious threat to global health. NS3 serine protease is one of the most advanced HCV drug targets. However, the high mutation rate makes many protease inhibitors ineffective and allows viral replication to continue. To investigate the structural basis of the molecular mechanism of HCV resistance to inhibitors, molecular dynamics and molecular mechanics Poisson–Boltzmann/surface area calculations were carried out on HCV NS3 serine protease–BI201335 complexes. The drug resistance to BI201335 is explained by the fact that seven single mutations weaken the biological activity by lessening the sum of electrostatic interactions in the gas phase and polar solvation. The computational results demonstrate that the mutations affect the BI201335 binding through direct and indirect mechanisms. Seven single mutations lead to significant changes in the conformation, such as the shifts of the side chain of His57 and Lys136 and the movement of the P2 group of BI201335 towards the solvent. Furthermore, the contributions of Lys136 significantly decrease, which is the most major binding attraction. The shifts of the side chain of His57 induce the lack of hydrogen bond between His57 with Asp81 expert for D168G mutation. Detailing the molecular mechanisms of BI201335 drug resistance provides some helpful insights into the nature of mutational effect and aid the rational design of potent inhibitors combating HCV.

QSAR and Docking Studies of HCV NS3 Serine Protease Inhibitors

Hepatitis C virus (HCV) is a Hepacivirus that causes chronic liver disease, leading to hepatocellular carcinoma, cirrhosis, and chronic hepatitis in about 3% of the world population. In this study, novel HCV NS3 serine protease inhibitors based on 93 boceprevir analogs were studied by QSAR analyses using thermodynamic, structural and topological descriptors, including E-state descriptors. Novel compounds were proposed using the QSAR models. Both models were highly predictive, with calibration, leave-one-out validation and external validation R2 of 0.66, 0.65 and 0.52, respectively. The most promising structures were docked into the HCV NS3 serine protease active site demonstrating, then, the high affinity of some new structures.

HCV Infection and NS-3 Serine Protease Inhibitors

Infection with Hepatitis C Virus (HCV) is a major health problem affecting 270 million people worldwide and 10 million people in Pakistan. Currently, there is no vaccine available for prevention of HCV infection due to the high degree of strain variation. The current standard of care is a combination of Pegylated interferon ? (PEG-INF ?) with ribavirin and Boceprevir/Telaprevir. Hence, there is a need to develop new antiviral agents from different targets. HCV NS3, a nonstructural protein has serine/protease and helicase domain, and these domains are considered as important antiviral drug targets to combat HCV. In the past 15 years, major scientific advances have enabled the development of many novel serine/protease and helicase inhibitors by using in vivo as well as in vitro model systems. Most of NS3 protease inhibitors have entered in different clinical trials phases but still there is a need to improve the functionality of these inhibitory compounds. This review summarizes potent antiviral compounds against HCV NS3 protease and their activity in combination with standard therapy. In conclusion, it will be very exciting to see HCV NS3-4A serine protease/helicase inhibitors progress through clinical developments and, hopefully, provide hepatitis C patients with much needed, more effective therapies.

Toward Second Generation Hepatitis C Virus NS3 Serine Protease Inhibitors: Discovery of Novel P4 Modified Analogues with Improved Potency and Pharmacokinetic Profile

Hepatitis C virus (HCV) infection is a global health crisis leading to liver cirrhosis, hepatocellular carcinoma, and liver failure in humans. Recently, we disclosed the discovery of Boceprevir, SCH 503034 (1), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that is currently undergoing phase III clinical trials. Our efforts toward a second generation HCV NS3 serine protease inhibitor were directed at improving the overall profile of the inhibitor. This article will elaborate on our studies leading to the discovery of new P4 modified inhibitors with enhanced potency and improved oral bioavailability. Thus, introduction of ether and carbamate-derived P4 moieties resulted in improving the replicon potency significantly. Incorporation of the P' secondary amide residue afforded significant improvement in pharmacokinetic properties. Combining the preferred moieties, identified from comprehensive SAR studies, resulted in inhibitors that displayed superior potency and very good oral as well as target organ exposure in rats.

Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Activity of compounds from Chinese herbal medicine Rhodiola kirilowii (Regel) Maxim against HCV NS3 serine protease

Treatment of the chronic hepatitis C virus (HCV) infection is an unmet medical need, and the HCV NS3 serine protease (NS3-SP) has been used as an attractive target of antiviral screening against HCV. To find naturally chemical entities as lead compounds from which novel anti-HCV agents could be developed, bioassay-guided fractionation and isolation were performed on a crude ethanol extract from rhizomes of the Chinese medicinal herb Rhodiola kirilowii (Regel) Maxim using column chromatography (CC) techniques and in vitro inhibitory activity against HCV NS3-SP. The partition of the extract between water and different organic solvents led to the isolation and identification of 12 compounds in the ethyl acetate part which proved to be the most active. These compounds were tested for in vitro activity against HCV NS3-SP, among which four (−)-Epicatechin derivatives: 3,3′-Digalloylproprodelphinidin B2 (Rhodisin, 1); 3,3′-Digalloylprocyanidin B2 ( 2); (−)-Epigallocatechin-3-O-gallate (EGCG, 3); and (−)-Epicatechin-3-O-gallate ( 4, ECG) represented the most potent ones with IC 50 of 0.77, 0.91, 8.51, and 18.55 μM, respectively. Salidroside, the commonly known compounds, together with the other compounds showed no activity up to 100.0 μM. Methylation and acylation of the hydroxyl groups of 1–4 caused a decrease of activity. Cell viability and secreted alkaline phosphatase (SEAP) activity assays with 1–4 revealed little if any toxicity. These nonpeptide inhibitors of HCV NS3-SP might serve as potential candidate anti-HCV agents.

Specific targeted antiviral therapy for hepatitis C

Since the discovery of the hepatitis C virus (HCV) as the major cause of non-A, non-B hepatitis in 1989, the search for specific targeted antiviral therapy for HCV (STAT-C) has been underway. Recently, major advances in the understanding of HCV biology and the development of an in vitro system of HCV replication have contributed to the selection of multiple candidate drugs for the treatment of hepatitis C. In 2006, five such candidate drugs have entered phase II clinical trials in patients chronically infected with hepatitis C, including small molecule inhibitors of the HCV NS3 serine protease and NS5B RNA-dependent RNA polymerase. This review focuses on hepatitis C protease and polymerase inhibitors that have progressed to phase II clinical development, foreshadowing the era of STAT-Cs.

SCH 503034, a Novel Hepatitis C Virus Protease Inhibitor, Plus Pegylated Interferon α-2b for Genotype 1 Nonresponders

SCH 503034 is a novel and potent oral hepatitis C virus (HCV) protease inhibitor. In this phase Ib study, we assessed safety parameters and virologic response of combination of SCH 503034 plus pegylated (PEG) interferon (IFN) alpha-2b in patients with HCV genotype 1 infections who were previously nonresponders to PEG-IFN-alpha-2b +/- ribavirin therapy. This was a multicenter, open-label, 2-dose level, 3-way crossover, randomized (to crossover sequence) study carried out in 3 medical centers in Europe. Adult patients received SCH 503034 200 mg (n = 14) or 400 mg (n = 12) 3 times daily orally and PEG-IFN-alpha-2b 1.5 microg/kg subcutaneously once each week. Patients received SCH 503034 as monotherapy for 1 week, PEG-IFN-alpha-2b as monotherapy for 2 weeks, and combination therapy for 2 weeks with washout periods between each treatment period. Combination therapy with SCH 503034 and PEG-IFN-alpha-2b was well tolerated, with no clinically significant changes in safety parameters. Mean maximum log(10) changes in HCV RNA were -2.45 +/- 0.22 and -2.88 +/- 0.22 for PEG-IFN-alpha-2b plus 200 mg and 400 mg SCH 503034, respectively, compared with -1.08 +/- 0.22 and -1.61 +/- 0.21 for SCH 503034 200 mg and 400 mg, respectively, and -1.08 +/- 0.22 and -1.26 +/- 0.20 for PEG-IFN-alpha-2b alone in the 200 mg and 400 mg SCH 503034 groups, respectively. SCH 503034 plus PEG-IFN-alpha-2b was well tolerated in patients with HCV genotype 1 nonresponders to PEG-IFN-alpha-2b +/- ribavirin. These preliminary results of antiviral activity of the combination suggest a potential new therapeutic option for this hard-to-treat, nonresponder patient population.

Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

In Vitro Studies of Cross-resistance Mutations against Two Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

In Vitro Resistance Studies of Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061: STRUCTURAL ANALYSIS INDICATES DIFFERENT RESISTANCE MECHANISMS

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.

Genetic screen for monitoring hepatitis C virus NS3 serine protease activity.

We have developed a genetic system to monitor the activity of the hepatitis C virus (HCV) NS3 serine protease. This genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a beta-galactosidase (betagal)-HCV NS3(2-181)/4(21-34) protease construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the betagal-HCV NS3(2-181)/4(21-34) protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS3(2-181)/4(21-34) protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.

Hepatitis C virus NS3 serine protease as a drug discovery target

Hepatitis C virus NS3 serine protease (HCV-Pr) is an extensively studied enzyme for drug intervention. The target presented serious challenges in early screening efforts, however, wish the lack of prominent active site features rendering traditional nonpeptidic serine protease inhibitor motifs and high-throughput screening campaigns ineffectual. In contrast, the peptidomimetic structure-based design approach has proven uccessful in the discovery of potent inhibitors of HCV Pr. Subsequent rational design efforts have led to the identification of an inhibitor that demonstrates efficacy in man, validating the years of research. This review summarizes why HCV Pr provides a viable drug discovery target despite the many obstacies, and details the breakthroughs in protein production and essay development that have facilitated inhibitor advances. The latest inhibitors in preclinical and clinical research and development are also presented, along with a discussion of how the recent HCV Pr clinical candidate chalienges much of the dogma surrounding peptidomimetic design. In addition, future issues such as resistance, genotype coverage and HIV-HCV coinfected individuals are considered.

A novel high throughput screening assay for HCV NS3 serine protease inhibitors

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.

Structure–activity relationship studies of a bisbenzimidazole-based, Zn 2+-dependent inhibitor of HCV NS3 serine protease

A survey of isosteric replacements of the phosphonoalanine side chain coupled with a process of conformational constraint of a bisbenzimidazole-based, Zn 2+-dependent inhibitor of hepatitis C virus (HCV) NS3 serine protease resulted in the identification of novel series of active compounds with extended side chains. However, Zn 2+-dependent HCV NS3 inhibition was relatively insensitive to the structural variations examined but dependent on the presence of negatively charged functionality. This result was interpreted in the context of an initial electrostatic interaction between protease and inhibitor that is subsequently consolidated by Zn 2+, with binding facilitated by the featureless active site and proximal regions of the HCV NS3 protein.