Identification of PTC725, an Orally Bioavailable Small Molecule That Selectively Targets the Hepatitis C Virus NS4B Protein

dThe hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors. Hepatitis C virus (HCV) is a significant public health threat, with up to 3% of the world population estimated to be harboring the infection. Although the virus can be naturally cleared, it more often becomes established as a chronic infection with an increased risk of poor long-term outcomes, including liver cirrhosis and cancer (1). Treatment of the disease historically used a combination of ribavirin and pegylated interferon, a cocktail with a 50% cure rate against genotype 1 but with a high likelihood of undesirable side effects (2, 3). The regulatory approval of directacting small-molecule antivirals represents a major advance in therapeutics by increasing the chance of efficacious treatment (4, 5), but there is still a need for novel antiviral therapies with reduced side effects and complementary resistance profiles. HCV inhibitors of the NS3 protease, of the multifunctional protein NS5A, and of the NS5B RNA-dependent RNA polymerase have been shown to be efficacious in the clinic, but inhibitors of NS4B have not been validated in vivo. We (6-8) and others (9-12) have described compounds targeting NS4B in vitro that, if developed, would offer a complementary mechanism that may improve the standard of care for hepatitis C virus infection treatment.HCV RNA replication occurs in vesicle-induced intracellular membranes derived from the cellular endoplasmic reticulum (ER), catalyzed by a complex of proteins encoded by the virus and host factors. NS4B is an integral membrane protein (13) that induces the rearrangement of intracellular membranes into a "membranous web," a collection of vesicles th...

Section: Gsk8853 Inhibits Multiple Hcv Genotypes and Generates Resistmentioning

confidence: 99%

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

Pouliot

Thomson

Xie

et al. 2015

“…The final DMSO concentration was 0.5% (vol/ vol). After 72 h of incubation, cells were harvested and subjected to realtime PCR analysis as reported previously (22,23). The primer/probe sets used for the PCR analysis on an ABI Prism 7900HTS sequence detection system are listed in Table S2 in the supplemental material.…”

Section: Compounds Grazoprevir {N-[[[(1r2r)-2-[5-(3-hydroxy-6-methomentioning

confidence: 99%

“…74104). RNA was then subjected to real-time PCR analysis as reported previously (22,23) using the above-mentioned primers and probes. The threshold cycle numbers (C T ) were normalized to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping gene to yield ⌬C T , and the log(1/power) (2,⌬C T Ϫ average ⌬C T of DMSO at day zero) was plotted over time using Prism (GraphPad Software Inc.).…”

Section: Compounds Grazoprevir {N-[[[(1r2r)-2-[5-(3-hydroxy-6-methomentioning

confidence: 99%

The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons

Lahser

Bystol

Curry

et al. 2016

Self Cite

The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC 90 ) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC 90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection. Hepatitis C virus (HCV) is a leading cause of chronic liver disease, with an estimated 130 to 170 million people infected globally. WHO estimates that more than 350,000 people die every year from hepatitis C-related liver diseases (1, 2, 3). The introduction of direct-acting antiviral agents (DAAs) as add-ons to the previous standard of care (SOC) consisting of pegylated interferon alpha plus ribavirin (PR) significantly improved sustained virologic response (SVR) rates from 40 to 50% to 65 to 70% in the previously hard-to-cure genotype 1 (GT1) patients after a 24-to 48-week treatment course. Further treatment advancements have been achieved with the introduction of interferon-free all-oral DAAs, with SVR rates now in excess of 90% after 12 weeks of therapy for GT1 patients (4, 5, 6). Recent reports indicate that therapy can be further simplified and likely shortened to Ͻ12 weeks in some cases while maintaining high SVR rates. Preexisting baseline resistance-associated variants (RAVs) and resistance selection remain contributory rea...

“…The cells were treated with inhibitor for 3 days, at which point they were washed with phosphate-buffered saline and lysed in cell lysis buffer (Ambion). The amount of replicon RNA level was measured by real-time quantitative PCR (TaqMan) assay (29,34). The PCR primers for GT1a replicons were located in the HCV internal ribosome entry site (IRES): IRES_F (5=-TGCGGAACCGGTGAGTACA-3=) and IRES_R (5=-GCGGGTTTATCCAAGAAAGGA-3=).…”

Section: Compounds Elbasvir Nn=-[[(6s)-6-phenyl-6h-indolomentioning

confidence: 99%

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons

Asante‐Appiah

Curry

McMonagle

et al. 2017

Self Cite

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n ϭ 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137-and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n ϭ 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.KEYWORDS HCV, genotype 4, resistance, antiviral, elbasvir, grazoprevir, NS5A, NS3/4A, antiviral agents, drug resistance mechanisms, hepatitis C virus H epatitis C virus (HCV) is a leading cause of chronic liver disease, with an estimated 170 million people infected globally (1, 2). The World Health Organization estimates that more than 350,000 people die every year from hepatitis C-related liver diseases (3). Approximately 20% of individuals chronically infected with HCV can be expected to develop liver cirrhosis and, of these, 6% will decompensate to end-stage liver disease, with an additional 4% developing hepatocellular carcinoma. A number of treatment options are now available with combinations of interferon-free direct-acting