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

Luciana Santos Pessoa,Amilcar Tanuri,Ana Luiza Chaves Valadão,Rodrigo Delvecchio Da Cunha,André Felipe Dos Santos,Celina Monteiro Abreu,Luãnna Liebscher Vidal,Emmerson C.B Da Costa

doi:10.1590/1678-4685-gmb-2016-0022

Abstract

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.

Highlights

It is estimated that 185 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) (Lin et al, 2004; Rice and Saeed, 2014)
The HCV genome consists of a single-stranded positivesense RNA of approximately 9.6 kb, which contains an open reading frame (ORF) encoding a polyprotein precursor of approximately 3.000 residues flanked by untranslated regions (UTRs) at both ends
HCV NS3 is a multifunctional protein in which the N-terminal constitutes a trypsin like protease and play a critical role in HCV processing by cleaving NS3 downstream at four sites

Summary

Introduction

It is estimated that 185 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) (Lin et al, 2004; Rice and Saeed, 2014). The HCV genome consists of a single-stranded positivesense RNA of approximately 9.6 kb, which contains an open reading frame (ORF) encoding a polyprotein precursor of approximately 3.000 residues flanked by untranslated regions (UTRs) at both ends. The precursor is cleaved into at least 10 different proteins: the structural proteins Core, E1, E2 and p7, as well as the non-structural proteins. HCV NS3 is a multifunctional protein in which the N-terminal constitutes a trypsin like protease and play a critical role in HCV processing by cleaving NS3 downstream at four sites (between NS3/NS4A, NS4A/NS4B, NS4B/NS5A, NS5A/NS5B). The carboxy-terminal region constitutes a superfamily 2 DExH/D-box RNA helicases that has NTPase activity (Bartenschlager et al, 2013). NS4A forms a stable complex with NS3 and is a co-factor for NS3 protease (Kim and Chang, 2013; Izquierdo et al, 2014)

Objectives

Methods

Results

Conclusion