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.

Hepatitis C: The Story of a Long Journey through First, Second, and Third Generation NS3/4A Peptidomimetic Inhibitors. What Did We Learn?

“…To address toxicity concerns (serum alanine transaminase (ALT) elevations), SAR exploration focused on the P1 vinyl-ACCA motif. This moiety, as seen, is a common structural feature of several PIs of previous generation (such as the linear inhibitors faldaprevir (32) and asunaprevir (38), and the macrocycles danoprevir (23), simeprevir (47), IDX320 (48), grazoprevir (52), and paritaprevir (65)). Unfortunately, it turned out to be toxic in clinical phases, due to its metabolic activation into a reactive epoxide probably reacting with nucleophile agents, like proteins and glutathione, and thus causing a transaminase increment.…”

“…Typically, the first fixed-dose combination of ledipasvir (2) (NS5A)/sofosbuvir (5) (NS5B) or ombitasvir (NS5A)/ paritaprevir (65) (NS3)/dasabuvir 39 (NS5B) (Figure 3 for ledipasvir (2) and sofosbuvir (5); Figure 23 for paritaprevir (65)) allowed to achieve >95% SVR. However, the first combination was limited by the high relapse rates, whereas the second one gave the possibility to discern patients with gt1a and gt1b and, in addition, was advantageous for patients with severe renal impairment.…”

“…The second wave first generation PIs included danoprevir (23) and vaniprevir (26) and resulted to be active against gt1, gt2, and gt4; they were still not effective against gt3. 62,65 All peptidomimetic drugs of first generation suffered from a broad cross-resistance due to Arg155 mutation. Moreover, mutations of Val36 and Thr54 were found to confer resistance to the covalent inhibitors of the first wave, while mutations of Asp168 affected the activity of macrocyclic and linear PIs of the second wave.…”

“…However, its oral systemic bioavailability was nondetectable in rat and monkey and in dog was around 41%. Evidence from in vitro metabolism data suggested that dual therapy with ritonavir could increase and sustain paritaprevir (65) concentrations in both plasma and liver in humans. However, limitations due to the restricted activity against other HCV genotypes were fulfilled with the next discovery and development of glecaprevir (74).…”

“…Limitations associated with the lack of efficacy of paritaprevir (65) against the gt3 variant 26 pushed the development of novel P2−P4 macrocycles, which culminated with the discovery of the potent pangenotypic of third generation glecaprevir (ABT-493, 74) (Figure 27). 36 This inhibitor originated from the lead compound 72 which showed high potency in replicon assay (EC 50 of 2.0 nM (gt1a), 2.9 nM (gt1b)) and improved metabolic stability in human liver microsomes (HLM Cl int of 31 μL/min/mg) compared to paritaprevir (65) (Cl int of 0.088 mL/min/kg).…”