Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A

Walker, Jill; Crosby, Renae M.; Wang, Amy; Woldu, Ermias; Vamathevan, Jessica; Voitenleitner, Christian; You, Shihyun; Remlinger, Katja; Duan, Maosheng; Kazmierski, Wieslaw M.; Hamatake, Robert

doi:10.1128/aac.01363-13

Cited by 23 publications

(24 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GSK8853 potency was shifted a relatively modest 3-fold for the PBC002 replicon compared to the standard genotype 1a replicon ( Table 1), suggesting that this virus could be used to test in vivo efficacy. A target of 14.1 ng/ml for an in vivo efficacious concentration was estimated by adjusting the EC 90 of the PBC002 replicon by 5.8-fold to account for binding to human serum proteins, as calculated by the change in EC 50 against genotype 1b stable replicons in the presence of 40% human serum (44,45). PXB mice were created by transplanting human hepatocytes into uPA ϩ/ϩ /SCID mice.…”

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

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

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...

show abstract

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

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…Replicon assays were performed as described previously (4). Briefly, stable-replicon-containing cell lines were seeded at a density of 2 ϫ 10 4 cells per well in 96-well assay plates in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin.…”

Section: Methodsmentioning

confidence: 99%

“…Stable cell lines carrying a bicistronic genotype 1a (H77), genotype 1b (Con1 strain with cell culture-adapted mutations) or genotype 2a (JFH-1) replicon were created in-house, licensed from ReBLikon GmbH (Mainz, Germany) or constructed in-house from HCVcc virus licensed from Apath, LLC (Brooklyn, NY), respectively (42,43). All three replicons express firefly luciferase and neomycin phosphotransferase and were maintained and propagated as described previously (4). ET-cured cells are a derivative of replicon-containing genotype 1b (Con1 strain) cells generated by treating Con1 cells with alpha interferon for several passages until HCV RNA levels were undetectable.…”

Section: Methodsmentioning

confidence: 99%

“…The standard of care for chronic HCV infection was a combination of pegylated alpha interferon and ribavirin up until the approval of the NS3 protease inhibitors telaprevir and boceprevir in 2011 (1,2). Other classes of DAAs showing clinical efficacy target the NS5A protein (3,4) or viral polymerase (NS5B; reviewed in reference 5). Of the other viral proteins, several smallmolecule DAAs to nonstructural protein NS4B have been identified (6)(7)(8)(9)(10)(11)(12)(13)(14), but to date, there are no examples of clinically active NS4B-targeting compounds.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Encoded Library Technology Screening of Hepatitis C Virus NS4B Yields a Small-Molecule Compound Series with In Vitro Replicon Activity

Zhu

Dickson

Parks

et al. 2015

Antimicrob Agents Chemother

View full text Add to dashboard Cite

To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC 50 s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 M against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets. Significant progress has been made in recent years in the discovery and development of novel direct-acting antivirals (DAAs) to treat hepatitis C virus (HCV) infection. However, the ability of HCV to rapidly evolve and develop resistance is such that there is a continued need to discover DAAs that act through novel mechanisms. The standard of care for chronic HCV infection was a combination of pegylated alpha interferon and ribavirin up until the approval of the NS3 protease inhibitors telaprevir and boceprevir in 2011 (1, 2). Other classes of DAAs showing clinical efficacy target the NS5A protein (3, 4) or viral polymerase (NS5B; reviewed in reference 5). Of the other viral proteins, several smallmolecule DAAs to nonstructural protein NS4B have been identified (6-14), but to date, there are no examples of clinically active NS4B-targeting compounds.NS4B is a small (27-kDa), hydrophobic, membrane-associated protein that is derived through processing of the HCV polyprotein by the viral protease NS3/4A (reviewed in references 15, 16, and 17). It complexes with other HCV nonstructural and possibly a host cell factor(s) to form the viral replicase (18-22), which serves to replicate the viral RNA and is likely coupled to the assembly process (20, 23). Besides its structural role in the replicase, NS4B is thought to be involved in the induction of membranous vesicles that provide a platform for HCV RNA replication (24, 25), and it may also participate in virion assembly and release (26,27). NS4B is also reported to hydr...

show abstract

“…JNJ‐56914845 (GSK2336805), the structure of which was disclosed by Kazmierski and colleagues, is a hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor in development for the treatment of chronic HCV infection (CHC). JNJ‐56914845 exerts potent activity against genotype 1a and 1b subtypes in HCV replicon systems in vitro, and a rapid, dose‐dependent antiviral response was observed following oral administration of single doses ranging from 1 to 120 mg to genotype 1 HCV‐infected patients . A clinical study of 4 weeks' administration of JNJ‐56914845 60 mg once daily in combination with pegylated interferon and ribavirin revealed that 73% of subjects had a rapid virological response, defined as undetectable HCV RNA in serum at 4 weeks of treatment .…”

mentioning

confidence: 99%

Pharmacokinetics of hepatitis C virus NS5A inhibitor JNJ‐56914845 (GSK2336805) in subjects with hepatic impairment

Adkison

Gan

Elko-Simms

et al. 2015

The Journal of Clinical Pharma

View full text Add to dashboard Cite

JNJ-56914845 (GSK2336805) is a hepatitis C virus nonstructural protein 5A inhibitor under development for the treatment of chronic hepatitis C (CHC) infection. This open-label, parallel-group, 2-part study evaluated the pharmacokinetics and safety of a single oral 60 mg dose of JNJ-56914845 in 4 cohorts: healthy, mild, moderate, and severe hepatic impairment (n = 8/cohort). Severity of hepatic impairment was categorized using Child-Pugh score, and the healthy subjects were matched for age, sex, body mass index, and smoking status to the moderate hepatic impairment cohort. JNJ-56914845 plasma AUC0-∞ was 26%, 52%, and 45% lower in subjects with mild, moderate, and severe hepatic impairment, respectively, relative to healthy subjects with no difference in half-life among the groups. The apparent oral clearance and volume of distribution were higher in subjects with hepatic impairment. The lower plasma concentrations were largely explained by decreased plasma protein binding in hepatically impaired subjects. One subject with severe hepatic impairment had 2 non-drug-related serious adverse events: an esophageal bleed requiring hospitalization, encephalopathy. Although hepatically impaired subjects have lower exposures than healthy matched controls, they had similar or slightly higher exposures than those observed in past studies of noncirrhotic, CHC patients, suggesting that no dose adjustments for hepatic impairment will be needed.

show abstract

Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A

Cited by 23 publications

References 51 publications

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

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

Encoded Library Technology Screening of Hepatitis C Virus NS4B Yields a Small-Molecule Compound Series with In Vitro Replicon Activity

Pharmacokinetics of hepatitis C virus NS5A inhibitor JNJ‐56914845 (GSK2336805) in subjects with hepatic impairment

Contact Info

Product

Resources

About