Cell transformation induced by hepatitis C virus NS3 serine protease

Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356–4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme.     

Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.     

GSK2878175, a pan‐genotypic non‐nucleoside NS5B polymerase inhibitor,in healthy and treatment‐naïve chronic hepatitis C subjects

GSK2878175 is a potent, pan‐genotypic, non‐nucleoside, nonstructural protein 5B palm polymerase inhibitor being developed for the treatment of chronic hepatitis C (CHC). A first‐in‐human, randomized, placebo‐controlled, dose escalation study, evaluated the safety and pharmacokinetics of GSK2878175 administered as single and repeat oral doses (once daily for 14 days) to healthy volunteers. A separate proof‐of‐concept, placebo‐controlled, repeat dose (once daily for 2 days) study evaluated the safety, pharmacokinetics and antiviral activity of GSK2878175 monotherapy in treatment‐naïve, noncirrhotic, subjects with hepatitis C virus (HCV) genotype 1 [1a and 1b], 2, or 3. No deaths or SAEs were reported in either study, and treatment was well‐tolerated. Across all the HCV genotypes, GSK2878175 monotherapy at doses of 10, 30 or 60 mg once daily for 2 days produced a statistically significant multilog reduction (P<.001) in plasma HCV RNA log₁₀ IU/mL from Baseline to 24, 48 and 72 hours after the first dose of GSK2878175 compared to placebo. The reduction in HCV RNA was sustained for a prolonged period across all of the active treatment groups, consistent with the long apparent half‐life of GSK2878175 that was observed (mean t_1/2 range: 60‐63 hours in the CHC subjects). In summary, GSK2878175, when administered to healthy subjects and subjects with CHC, did not reveal any safety concerns that would limit or preclude further clinical development. GSK2878175 monotherapy across a wide dose range produced substantial reduction in HCV RNA, irrespective of HCV genotype. The results from these studies support further evaluation of GSK2878175‐based regimens.     

Hepatitis C virus resistance to protease inhibitors

Recent advances in molecular biology have led to the development of novel small molecules that target specific viral proteins of the hepatitis C virus (HCV) life cycle. These drugs, collectively termed directly acting antivirals (DAA) against HCV, include a range of non-structural (NS) 3/NS4A protease, NS5B polymerase, and NS5A inhibitors at various stages of clinical development. The rapid replication rate of HCV, along with the low fidelity of its polymerase, gives rise to generations of mutations throughout the viral genome resulting in remarkable sequence variation in the HCV population, known as a quasispecies. The efficacy of DAAs is limited by the presence of those mutations that give rise to amino-acid substitutions within the targeted protein, and that affect the viral sensitivity to these compounds. Thus, due to the high genetic variability of HCV, variants with reduced susceptibility to DAA can occur naturally even before treatment begins, but usually at low levels. Not surprisingly then, these changes are selected in patients either breaking through or not responding to potent DAA treatment. In vitro or in vivo, six major position mutations in the NS3 HCV protease (36, 54, 155, 156, 168, and 170) have now been reported associated with different levels of resistance. The amino acid composition at several of the drug resistance sites can vary between the HCV genotypes/subtypes, resulting in different consensus amino acids leading to a reduction in replicative fitness as well as reduced DAA sensitivity. Different amino acid diversity profiles for HCV genotypes/subtypes suggest differences in the position/type of immune escape and drug resistance mutations. Also, different pathways of resistance profiles based on the chemical scaffold (linear or macrocyclic) of the protease inhibitors have been described. This review first describes how resistance to a protease inhibitor can develop and then provides an overview of the mechanism of how particular mutations confer varying levels of resistance to protease inhibitor, which have been identified and characterized using both genotypic and phenotypic tools. Future potential therapeutic strategies to assist patients who do develop resistance to protease inhibitors are also outlined. The challenge developing new HCV protease inhibitors should take into consideration not only the antiviral potency of the drugs, the occurrence and importance of side effects, the frequency of oral administration, but also the resistance profiles of these agents.     

Sofosbuvir plus ribavirin in treatment‐naïve patients with chronic hepatitis C virus genotype 1 or 3 infection in India

Until 2014, pegylated interferon plus ribavirin was the recommended standard of care for the treatment of chronic hepatitis C virus (HCV) infection in India. This open‐label phase 3b study, conducted across 14 sites in India between 31 March 2014 and 30 November 2015, evaluated the efficacy and safety of sofosbuvir plus ribavirin therapy among treatment‐naïve patients with chronic genotype 1 or 3 HCV infection. A total of 117 patients with genotype 1 or 3 HCV infection were randomized 1:1 to receive sofosbuvir 400 mg and weight‐based ribavirin (1000 or 1200 mg) daily for 16 or 24 weeks. Among those with genotype 1 infection, the primary efficacy endpoint of sustained virologic response at 12 weeks post‐treatment (SVR12) was reported in 90% (95% confidence intervals [CI], 73‐98) and 96% (95% CI, 82‐100) of patients following 16 and 24 weeks of treatment, respectively. For patients with genotype 3 infection, SVR12 rates were 100% (95% CI, 88‐100) and 93% (95% CI, 78‐99) after 16 and 24 weeks of therapy, respectively. Adverse events, most of which were mild or moderate in severity, occurred in 69% and 57% of patients receiving 16 and 24 weeks of treatment, respectively. The most common treatment‐emergent adverse events were asthenia, headache and cough. Only one patient in the 24‐week group discontinued treatment with sofosbuvir during this study. Overall, sofosbuvir plus ribavirin therapy achieved SVR12 rates ≥90% and was well tolerated among treatment‐naïve patients with chronic genotype 1 or 3 HCV infection in India.     

Antiviral response and resistance analysis of treatment‐naïve HCV‐infected patients receiving single and multiple doses of GS‐9190

GS‐9190 is a NS5B non‐nucleoside analogue with demonstrated effectiveness in a Phase 1 monotherapy study and in combination with other DAAs for treatment of chronic HCV infection. Here, the resistance profile of GS‐9190 monotherapy in a Phase 1b study was investigated. Resistance analysis was performed by population sequencing and allele‐specific PCR (AS‐PCR) for Y448H with an assay cut‐off of 0.5%. Phenotypic susceptibility analyses were performed on patient isolates as well as site‐directed mutagenesis of mutations selected during monotherapy. No resistance‐associated variants were observed in patients before or after receiving single doses of GS‐9190 by population sequencing. In contrast, in patients who received GS‐9190 for 8 days, mutations Y448H and Y452H in NS5B were observed by population sequencing in 21/36 (58%) and 2/36 (5.6%) patients, respectively, at Day 8 or Day 14. Among the remaining 15 patients who had no detectable Y448H at Day 8 or Day 14 by population sequencing, low frequencies of Y448H ranging from 1.3 to 9.7% were detected in 14 of 15 patients by AS‐PCR. By AS‐PCR, Y448H remained detectable at reduced frequency in the majority of patients analysed through 4–6 months of follow‐up. Chimeric HCV replicons constructed with the NS5B sequence from patients with Y448H and Y448H + Y452H/Y demonstrated 27‐fold and 78.5‐fold reduced susceptibility to GS‐9190. In conclusion, Y448H was rapidly selected in the majority of patients receiving multiple doses of GS‐9190 as monotherapy, despite undetectable levels in pretreatment samples. Y448H confers reduced susceptibility to GS‐9190 and other NNIs and persisted in most patients for months post‐treatment.     

Evolutionary dynamics of hepatitis C virus NS3 protease domain during and following treatment with narlaprevir,a potent NS3 protease inhibitor

Narlaprevir, a hepatitis C virus (HCV) NS3/4A serine protease inhibitor, has demonstrated robust antiviral activity in a placebo‐controlled phase 1 study. To study evolutionary dynamics of resistant variants, the NS3 protease sequence was clonally analysed in thirty‐two HCV genotype 1–infected patients following treatment with narlaprevir. Narlaprevir monotherapy was administered for one week (period 1) followed by narlaprevir/pegylated interferon‐alpha‐2b combination therapy with or without ritonavir (period 2) during two weeks, interrupted by a washout period of one month. Thereafter, all patients initiated pegylated interferon‐alpha‐2b/ribavirin combination therapy. Longitudinal clonal analysis was performed in those patients with NS3 mutations. After narlaprevir re‐exposure, resistance‐associated mutations at position V36, T54, R155 and A156 were detected in five patients in >95% of the clones. Narlaprevir retreatment resulted in a 2.58 and 5.06 log₁₀ IU/mL viral load decline in patients with and without mutations, respectively (P = <0.01). After treatment, resistant variants were replaced with wild‐type virus within 2–24 weeks in three patients. However, the R155K mutation was still observed 3.1 years after narlaprevir dosing in two patients in 5% and 45% of the viral population. Resistant variants could be detected early during treatment with narlaprevir. A slower viral load decline was observed in those patients with resistance‐associated mutations detectable by direct population sequencing. These mutations disappeared within six months following treatment with the exception of R155K mutation, which persisted in two patients.     

GS‐9857 in patients with chronic hepatitis C virus genotype 1–4 infection: a randomized,double‐blind,dose‐ranging phase 1 study

GS‐9857, an inhibitor of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A, demonstrates potent activity against HCV genotypes 1–6 and improved coverage against commonly encountered NS3 resistance‐associated variants (RAVs). In this study, the safety, tolerability, antiviral activity and pharmacokinetics (PK) of GS‐9857 were evaluated in patients with chronic HCV genotype 1–4 infection. Patients with genotype 1–4 infection received placebo or once‐daily GS‐9857 at doses ranging from 50 to 300 mg for 3 days under fasting conditions. GS‐9857 was well tolerated; all reported adverse events (AEs) were mild or moderate in severity. Diarrhoea and headache were the most commonly reported AEs. Grade 3 or 4 laboratory abnormalities were observed in 17% of patients receiving GS‐9857; there were no Grade 3 or 4 abnormalities in alanine aminotransferase, aspartate aminotransferase or alkaline phosphatase levels. GS‐9857 demonstrated potent antiviral activity in patients with chronic HCV infection, achieving mean and median maximum reductions in HCV RNA of ≥3 log₁₀ IU/mL following administration of a 100‐mg dose in patients with HCV genotype 1a, 1b, 2, 3 or 4 infection. The antiviral activity of GS‐9857 was unaffected by the presence of pretreatment NS3 RAVs. In patients with genotype 1–4 infection, GS‐9857 exhibited linear PK and was associated with a median half‐life of 29–42 h, supporting once‐daily dosing. Thus, the tolerability, efficacy and pharmacokinetic profile of GS‐9857 support its further evaluation for treatment of patients with chronic HCV infection.     

Simeprevir plus sofosbuvir for eight or 12 weeks in treatment‐naïve and treatment‐experienced hepatitis C virus genotype 4 patients with or without cirrhosis

The OSIRIS study investigated efficacy and safety of simeprevir plus sofosbuvir for eight or 12 weeks in hepatitis C virus (HCV) genotype 4‐infected patients with METAVIR F0‐F4 fibrosis. Sixty‐three patients (33 treatment‐naïve and 30 peg‐interferon/ribavirin (Peg‐IFN/RBV)‐experienced) enrolled in a partly randomized, open‐label, multicentre, phase IIa study. Patients with F0‐F3 fibrosis were randomized (1:1) into two groups (A1 and A2), stratified according to treatment experience and METAVIR score, to receive either eight weeks (Group A1, n=20) or 12 weeks (Group A2, n=20) of treatment. Patients with compensated cirrhosis (METAVIR F4) received 12 weeks of treatment (Group B, n=23). Treatment comprised simeprevir 150 mg and sofosbuvir 400 mg daily. The primary efficacy endpoint was sustained virologic response 12 weeks after planned end of treatment (SVR12). Safety and tolerability were assessed throughout. Overall, 92% (95% CI: 82‐97) of patients achieved SVR12; 75% (15/20) in Group A1 and 100% in groups A2 and B. Patients who did not achieve SVR12 (n=5) experienced viral relapse during the first 32 days following treatment and were all prior Peg‐IFN/RBV null responders. The most commonly reported treatment‐emergent adverse events (TEAEs) were asymptomatic lipase increase (14%), pruritus (14%), headache (13%) and hyperbilirubinaemia (11%). No patients discontinued due to TEAEs. In conclusion, simeprevir plus sofosbuvir for 12 weeks achieved a 100% SVR rate in HCV genotype 4‐infected patients with or without compensated cirrhosis (ClinicalTrials.gov: NCT02278419). The AE and laboratory profile were favourable and consistent with previous data for simeprevir plus sofosbuvir in eight‐ and 12‐week regimens.     

丙型肝炎病毒NS3蛋白酶结构域在昆虫细胞中的表达及鉴定

目的　利用杆状病毒 昆虫细胞表达系统制备丙型肝炎病毒 (HCV)丝氨酸蛋白酶结构域 (proteasedomain)的重组蛋白。方法　构建含有HCV丝氨酸蛋白酶结构域基因的杆状病毒转移载体 pFBCNS3N ,转化大肠杆菌DH10Bac进行转座 ,提取重组Bacmid用Lipofectamine法转染昆虫细胞Sf9包装成重组杆状病毒。用重组杆状病毒感染昆虫细胞进行蛋白表达 ,用SDS PAGE电泳和免疫印迹实验分析表达情况 ;利用去垢剂十二烷基肌酸钠溶解重组蛋白后以Ni NTAagrose柱亲和纯化重组蛋白 ;以重组蛋白NS5ab(包含NS5A羟基端部分和NS5B氨基端部分的重组蛋白 )为底物检测丝氨酸蛋白酶的酶切活性 ;以间接ELISA法分析重组蛋白的抗原性。结果　HCV丝氨酸蛋白酶结构域基因在昆虫细胞中获得高水平表达 ;重组蛋白能够部分溶解于含有去垢剂十二烷基肌酸钠的缓冲液 ;从 5× 10 7细胞中总共获得 0 .2mg重组蛋白 ,纯度大于 90 %;重组丝氨酸蛋白酶能够将NS5ab切割成两个片段 ;血清学实验证实该蛋白抗原性很好。结论　昆虫细胞表达的HCV丝氨酸蛋白酶结构域为膜结合型蛋白 ,具有良好的酶学活性以及抗原性。     

A phase 1, randomized,dose‐ranging study of GS‐5816, a once‐daily NS5A inhibitor,in patients with genotype 1–4 hepatitis C virus

GS‐5816 is an inhibitor of the hepatitis C virus (HCV) NS5A protein that has demonstrated pan‐genotypic activity and a high barrier to resistance in HCV replicon assays. The aim of this study was to evaluate the safety, antiviral activity and pharmacokinetics of once‐daily doses of GS‐5816 in patients with genotype 1–4 HCV infection. Patients with genotype 1–4 HCV infection were randomized to 3 days of GS‐5816 at doses ranging from 5 to 150 mg or placebo. Adverse events were recorded, and plasma samples obtained for analysis of pharmacokinetics, HCV RNA and NS5A sequencing studies. GS‐5816 5–150 mg for 3 days was well tolerated and resulted in rapid declines in HCV RNA that were sustained over the dosing period. In patients treated with the 150 mg dose of GS‐5816, the mean maximal HCV RNA declines were 4.0, 4.0, 4.4, 3.3 and 3.5 log₁₀ IU/mL in patients with genotype 1a, 1b, 2, 3 and 4 HCV infection, respectively. Pretreatment NS5A resistance‐associated polymorphisms were detected in 31% (22/70) of patients. Genotype 1 and 3 HCV‐infected patients without pretreatment NS5A resistance‐associated polymorphisms had greater declines in HCV RNA than patients with resistance‐associated polymorphisms. Plasma pharmacokinetics were supportive of once‐daily dosing. GS‐5816 demonstrated pangenotypic antiviral activity in patients with genotype 1‐4 HCV infection. It will be further evaluated in combination with other pangenotypic direct‐acting antivirals to achieve the goal of developing a well‐tolerated, highly effective treatment for all HCV genotypes.     

Efficacy of 12 or 18 weeks of elbasvir plus grazoprevir with ribavirin in treatment‐naïve,noncirrhotic HCV genotype 3‐infected patients

Elbasvir (EBR; HCV NS5A inhibitor) and grazoprevir (GZR; HCV NS3/4A protease inhibitor) are approved as a fixed‐dose combination to treat patients chronically infected with HCV genotypes 1 and 4. During the development programme and supported by in vitro potency, the efficacy of EBR+GZR was assessed in HCV GT3‐infected patients. This study's aim was to determine the efficacy and tolerability of 12 or 18 weeks of EBR+GZR with ribavirin (RBV) in treatment‐naïve, noncirrhotic HCV GT3‐infected patients. Randomized patients received open‐label EBR (50 mg once daily) + GZR (100 mg once daily) + RBV. The primary efficacy objective was to evaluate the sustained virologic response rates 12 weeks after the end of all study therapy (SVR12). SVR12 rates (95% confidence interval) were 45.0% (23.1, 68.5) and 57.1% (34.0, 78.2) after treatment with EBR+GZR+RBV for 12 weeks or 18 weeks, respectively. On‐treatment virologic failure was observed in 41% (17 of 41) of patients. At virologic failure, resistance‐associated substitutions (RASs) with a >five‐fold shift in potency occurred in the NS3 region in six (35%) patients and in the NS5A region in 16 (94%) patients. The most common RAS at virologic failure was Y93H in NS5A which was identified in 13 of 17 (76%) patients. The efficacy of EBR+GZR+RBV was suboptimal in HCV GT3‐infected patients due to a high rate of on‐treatment virologic failure and treatment‐emergent RASs which demonstrates an inadequate barrier to the development of GT3 resistance. However, rapid viral clearance demonstrated the antiviral activity of EBR+GZR+RBV in GT3‐infected patients.clinicaltrials.gov: NCT01717326.     

Natural prevalence of HCV minority variants that are highly resistant to NS3/4A protease inhibitors

Summary. Minority drug‐resistant hepatitis C virus (HCV) variants may go undetected yet be clinically important. NS3/4A protease resistance substitutions V36A and A156S/T/V were selected in patients treated with protease inhibitors. The aim of this study was to investigate whether these substitutions pre‐existed in HCV infected patients. An allele‐specific PCR protocol that detected the NS3/4A protease resistance substitutions V36A and A156S/T/V was used to determine the prevalence of naturally occurring variants in 45 patients. All patient samples were infected with HCV of genotype 1b and were naïve for pegIFNα/ribavirin treatment. Thirty samples (67%) had at least one HCV PI‐resistant variant. A156T (23, 51%) was detected more frequently than A156V (13, 29%) or A156S (1, 2%). V36A was detected in 12 samples (27%). These results demonstrate the high prevalence of minority drug‐resistant NS3/4 protease resistance substitutions. Our results also demonstrate that allele‐specific PCR can be used to detect minor HCV NS3 protease resistant variants in pretreatment samples and to study in detail the evolution of mutant viruses during targeted antiviral therapy.