Hepatitis C Replication Inhibitors That Target the Viral NS4B Protein

Miller, John F.; Chong, Pek Y.; Shotwell, J. Brad; Catalano, John G.; Tai, Vincent W.‐F.; Fang, Jing; Banka, Anna L.; Roberts, Christopher D.; Youngman, Michael; Zhang, Huichang; Xiong, Zhiping; Mathis, Amanda; Pouliot, Jeffery J.; Hamatake, Robert; Price, Daniel J.; Seal, John W.; Stroup, Lisa L.; Creech, Katrina L.; Carballo, Luz H.; Todd, Dan; Spaltenstein, Andrew; Furst, Sylvia M.; Hong, Zhi; Peat, Andrew J.

doi:10.1021/jm400125h

Cited by 45 publications

(47 citation statements)

References 71 publications

(104 reference statements)

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“…We therefore used a humanized-mouse model to determine the efficacy of GSK8853 as monotherapy to treat HCV infection with a genotype 1a PBC002 virus strain. Abbreviated results from this study along with data on the synthesis of prodrug GSK9574 have been previously reported (6), and this current work significantly expands on the previously reported data and analysis. Prior to compound testing performed with the in vivo infection model, the NS4B sequence from PBC002 was used to construct a chimeric replicon to ensure that the potency of GSK8853 against this strain was similar to that of our standard in vitro replicons.…”

Section: Gsk8853 Inhibits Multiple Hcv Genotypes and Generates Resistsupporting

confidence: 76%

“…The previously described preclinical drug metabolism and pharmacokinetics (DMPK) data for GSK8853 (7) suggested that an efficacious dose would not be easily attainable, so a phosphate prodrug form of this compound with improved DMPK properties, GSK9574 (Fig. 1B), was selected for use (6). A pilot PK study of GSK9574 in PXB mice determined that oral BID dosing at 10, 30, or 100 mg/kg would deliver levels of GSK8853 expected to be efficacious (see Fig.…”

Section: Gsk8853 Inhibits Multiple Hcv Genotypes and Generates Resistmentioning

confidence: 99%

“…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)(7)(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.…”

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confidence: 99%

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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

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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...

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Section: Gsk8853 Inhibits Multiple Hcv Genotypes and Generates Resistsupporting

confidence: 76%

Section: Gsk8853 Inhibits Multiple Hcv Genotypes and Generates Resistmentioning

confidence: 99%

mentioning

confidence: 99%

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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

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“…26 Since its introduction, MFSDA has been used as a convenient source of 'CuCF 3 ' and has proved useful in the trifluoromethylation of subpophyrins, 27 pyrazoles, 28 purine nucleosides, 29,30 oxazolyl intermediates, 31 pyrazolopyridines, 32 and others. 33,34 The accepted mechanism comprises an initial step involving the formation of a copper salt with the elimination of methyl halide.…”

Section: A N U S C R I P Tmentioning

confidence: 99%

Access to trifluoromethylated 4-alkoxy-2-pyrones, pyridones and quinolones

Clarke

McGlacken

2015

Tetrahedron

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“…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.…”

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confidence: 99%

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

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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...

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Hepatitis C Replication Inhibitors That Target the Viral NS4B Protein

Cited by 45 publications

References 71 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

Access to trifluoromethylated 4-alkoxy-2-pyrones, pyridones and quinolones

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

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