Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate

Han, Bin; Dvory‐Sobol, Hadas; Greenstein, Andrew E.; McCarville, Joseph; Hung, Magdeleine; Liu, Xiaohong; Miller, Michael D.; Mo, Hongmei

doi:10.1016/j.jviromet.2015.09.002

Cited by 3 publications

(3 citation statements)

References 61 publications

(46 reference statements)

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“…Then, they were discontinued and Simeprevir was incorporated in the protocol along with Sofosbuvir and Datasclavir. IC 50 values obtained for NS3 WT were in accordance to previously reported data ( Sarrazin et al , 2007 ; Jiang et al , 2013 ; Izquierdo et al , 2014 ; Han et al , 2015 ).…”

Section: Discussionsupporting

confidence: 92%

“…Some HCV mutations or chimeras encoding sequences from patients isolates have significant low replication capacity. In addition, replicons generation is an expensive and time consuming method ( Han et al. , 2015 ).…”

Section: Discussionmentioning

confidence: 99%

“…Since the replicon system was developed, enzymatic assays were hardly used for drug screening. Recently, Han and co-workers demonstrated a fast and reproducible assay to measure NS3 activity, emphasizing that this kind of research can be very useful for clinical samples ( Han et al , 2015 ). It is worth noting that, in Brazil, there is no phenotypic test available to predict HCV mutants susceptibility to the current treatment, and this present work provides strong evidences for its feasibility and implementation.…”

Section: Discussionmentioning

confidence: 99%

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Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

et al. 2016

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Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

et al. 2016

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NS3 protease resistance-associated substitutions in liver tissue and plasma samples from patients infected by hepatitis C virus genotype 1A or 1B

et al. 2017

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The presence of naturally occurring resistance-associated substitutions (RASs) in the HCV-protease domain has been poorly investigated in the liver, the main site of HCV replication. We evaluated the natural resistance of the virus to NS3 protease inhibitors in liver tissue and plasma samples taken from HCV-infected patients. RASs were investigated by means of viral population sequencing in liver tissue samples from 18 HCV-infected patients harbouring genotype 1a or genotype 1b; plasma samples from 12 of these patients were also available for virological investigation. A discordant genotype was found in two of the 12 patients (16.6%) who provided samples from both compartments. Sequence analysis of the NS3 protease domain showed the presence of RASs in four of the 18 liver tissue samples (22.2%), two of which showed cross-resistance to protease inhibitors in clinical use or phase 2-3 trials. The analysis of the 12 paired tissues and plasma samples excluded the presence of RASs in the plasma compartment. The dominance of discordant genotypes in the paired liver and plasma samples of some HCV-infected patients suggests mixed infection possibly leading to the selective advantage of different genotype in the two compartments. The presence of RASs at intra-hepatic level is not uncommon and may lead to the early emergence of cross-resistant strains.

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Real-world challenges for hepatitis C virus medications: a critical overview

Shahid

Almalki

Hassan

et al. 2017

Critical Reviews in Microbiology

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From 2010, the landscape of hepatitis C therapeutics has been changed rapidly, and today we are standing at a cusp of a pharmacological revolution where highly effective and interferon (IFN)-free direct acting antivirals (DAAs) are already on the market. Such treatment paradigms attain 90-95% sustained virologic response (SVR; undetectable viral load at week 12 or 24 at the end of therapy) rates in treated individuals compared to 50-70% with treatment completion of dual-therapy-pegylated interferon (PEG-IFN) and ribavirin (RBV). As the major goal now for the hepatologists, clinicians, physicians, and health care workers is likely to eradicate hepatitis C infection in parallel to treatment, the demand is for a one-size-fits-all pill that could be prescribed beyond the limitations of hepatitis C genotype, viral load, previous treatment history, advanced hepatic manifestations (fibrosis, cirrhosis) and antiviral drug resistance. Although the new treatment strategies have shown high cure rates in clinical trials, such treatment paradigms are posing dilemmas too in real-world clinical practice. Therapy cost, treatment access to low and middle-income countries, treatment-emergent adverse events, lack of effective viral screening and disease progression simulation models are potential challenges in this prospect. This review article deeply overviews the challenges encountered while surmounting the burden of hepatitis C around the world.

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Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate

Cited by 3 publications

References 61 publications

Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

NS3 protease resistance-associated substitutions in liver tissue and plasma samples from patients infected by hepatitis C virus genotype 1A or 1B

Real-world challenges for hepatitis C virus medications: a critical overview

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