A small molecule inhibits HCV replication and alters NS4B's subcellular distribution

Bryson, Paul D.; Cho, Nam‐Joon; Einav, Shirit; Lee, Choong-Ho; Tai, Vincent W.‐F.; Bechtel, Jill; Sivaraja, Mohan; Roberts, Chris; Schmitz, Uli; Glenn, Jeffrey S.

doi:10.1016/j.antiviral.2010.03.013

Cited by 42 publications

(40 citation statements)

References 25 publications

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“…These results indicate that the NS4B inhibitors impair NS4B function related to viral replication without interrupting its subcellular localization. Interestingly, the NS4B inhibitor anguizole, shown to interact with the AH2 domain of NS4B, was reported to alter the subcellular distribution of NS4B-green fluorescent protein (GFP) fusion protein (52). The difference in results for our NS4B inhibitors compared to those for anguizole suggests that these agents have different mechanisms of action in affecting NS4B function and subcellular localization.…”

Section: Discussioncontrasting

confidence: 45%

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

Graci

Lahser

et al. 2013

Antimicrob Agents Chemother

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While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC 50 ) of 1.7 nM and an EC 90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.C hronic hepatitis C virus (HCV) infection is a worldwide epidemic disease with an estimate of over 170 million people chronically infected worldwide (1). Approximately 60 to 85% of HCV infections result in chronic hepatitis that can lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma (2). The current standard of care (SOC) for chronic hepatitis C infection, pegylated alpha interferon in combination with ribavirin, has serious side effects and limited efficacy, especially for infection with HCV genotype 1, which is the most prevalent HCV genotype (3, 4). Two HCV protease inhibitors, boceprevir (Victrelis) and telaprevir (Incivek), for the therapy of HCV genotype 1 infection in combination with the SOC were approved for use nearly 2 years ago. In addition, a number of other direct-acting antivirals (DAAs) in clinical trials have demonstrated encouraging efficacy in combination therapies (5). Currently, the HCV antivirals in preclinical and clinical development are inhibitors of the viral protease, polymerase, or nonstructural protein 5A (NS5A) (6). Due to the emergence of viral variants resistant to the DAAs, even in combination therapy with the SOC (7-9) and the potential for viral rebound after cessation of antiviral therapy, it is essential to discover and develop novel HCV inhibitors that act on new targets and can be used in combination with the SOC and/or DAAs to enhance efficacy and to delay or possibly prevent the emergence of drug-resistant...

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Section: Discussioncontrasting

confidence: 45%

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

Graci

Lahser

et al. 2013

Antimicrob Agents Chemother

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“…Analysis of the genotype 1b replicon sequence after selection revealed changes from the wild type that resulted in amino acid changes H94R, F98L, and V105M, similarly to reports of studies performed with other NS4B inhibitors (7,11,12). These sequence changes were inserted into parental replicons and tested for GSK8853 sensitivity.…”

Section: Gsk8853 Inhibits Multiple Hcv Genotypes and Generates Resistmentioning

confidence: 99%

“…Previous reports have identified small-molecule binders of NS4B that are associated with inhibition of particular biochemical functions. The reported RNA binding of NS4B can be inhibited by clemizole, a weak inhibitor of HCV replication (25), while a pyrazolopyrimidine compound referred to as anguizole was able to disrupt dimerization of NS4B protein (21) and cause intracellular rearrangements of NS4B protein (12). Both anguizole and an unrelated amiloride compound have also been reported to affect the association of membrane vesicles with NS4B peptides in vitro (26).…”

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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

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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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“…The N terminus of NS4B contains a predicted amphipathic ␣-helix (AH1 within the first 27 amino acids of NS4B) (15, 19) and a structurally resolved amphipathic ␣-helix (AH2, spanning amino acids 42 to 66) (19). AH2 contributes to NS4B association with membranes (19), is a major determinant for NS4B oligomerization (21), and plays an important role in HCV RNA replication (8,9,19). The C-terminal domain extends from amino acids 191 to 261 of NS4B and comprises arginine residues important for RNA binding of NS4B (14), a predicted ␣-helix (H1, residues 201 to 212) (20,25,43), a nucleotidebinding motif (228 to 231) (42), a membrane-associated amphipathic ␣-helix (H2, residues 229 to 253) (20), and two palmitoylation sites at the very C terminus (45).…”

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confidence: 99%

Charged Residues in Hepatitis C Virus NS4B Are Critical for Multiple NS4B Functions in RNA Replication

Blight

2011

J Virol

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The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N-and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex.Hepatitis C virus (HCV) is enveloped with a single-stranded positive-sense RNA genome. The HCV genome is about 9,600 nucleotides in length and encodes a single polyprotein that is processed by cellular and viral proteases into three structural proteins (core, E1, and E2), a small ion-channel protein (p7), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV RNA replication requires the NS3, NS4A, NS4B, NS5A, and NS5B proteins, and biochemical functions have been well studied for NS3, NS4A, and NS5B (reviewed in references 3, 7, and 30). The NS3 protein contains the serine protease activity responsible for cleavages at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions of the viral polyprotein, as well as the RNA helicase/NTPase activities essential for RNA replication. NS4A forms a stable complex with NS3, functions as a cofactor for the enzymatic activities of NS3, and anchors NS3 to intracellular membranes. The NS5B protein is the RNA-dependent RNA polymerase responsible for synthesizing the positive-sense RNA genome via negativestrand intermediates. While the integral endoplasmic reticulum (ER) membrane protein NS4B and the phosphorylated NS5A protein are essential for RNA replication, their functions are not completely understood.The NS4B protein is predicted to comprise N-and C-termi...

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A small molecule inhibits HCV replication and alters NS4B's subcellular distribution

Cited by 42 publications

References 25 publications

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

Identification of PTC725, an Orally Bioavailable Small Molecule That Selectively Targets 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

Charged Residues in Hepatitis C Virus NS4B Are Critical for Multiple NS4B Functions in RNA Replication

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