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1.  Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿ 
GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain.
doi:10.1128/AAC.00307-11
PMCID: PMC3165336  PMID: 21746939
2.  Optimized high-throughput screen for Hepatitis C virus translation inhibitors 
Journal of Biomolecular Screening  2011;16(2):211-220.
Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. We developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5′ capped mRNA control. We screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1,700 initial hits. After secondary counter screening the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end we found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, and the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.
doi:10.1177/1087057110391665
PMCID: PMC3260011  PMID: 21297107
Hepatitis C virus (HCV); IRES; luciferase; high-throughput screen; rabbit reticulocyte lysate
3.  Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a▿  
The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (∼70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.
doi:10.1128/AAC.00289-10
PMCID: PMC2916306  PMID: 20516274

Results 1-3 (3)