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

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.

Figure 1. NS3 purification. (A) SDS-PAGE of the eluted fractions of NS3 WT (lanes 1-6). Lane 7 corresponds to pellet resuspension and lane 8 to the flowthrough, collected after the supernatant went through the nickel agarose resin. (B) Western blot of two mutants (V170I and T54S + V170I) showing purified proteins in imidazol elution fractions, stained with anti-HIS antibody.

Figure 2. Exponential curve obtained from the raw data analysis of the enzymatic inhibition reaction with nine concentrations in μM of Simeprevir and NS3 WT. Readings were made in every 30 seconds for 50 minutes. These conditions were used for all enzymes evaluated. The initial velocity analysis was obtained from this graphic to calculate IC₅₀ values and linear regression curve.

Figure 3. Determination of the IC50 values of three NS3 protease inhibitors (Telaprevir - TVP; Boceprevir - BCP and Simeprevir - SMP) in relation to NS3 WT. IC₅₀ for TVP was 0.13 μM, for BCP it was 0.258 μM, and for SMP it was 0.017 μM. These values were established by sigmoidal non linear regression logistic 4-parameter analysis using Sigma Plot software.

Figure 4. Determination of the median fold change values for three NS3 protease inhibitors (Telaprevir; Boceprevir and Simeprevir) for the five mutants evaluated. IC₅₀ median values obtained for each mutant using the PIs were used to calculate the fold change median (mutants IC50 median/WT IC50 median).