Plural assay systems derived from different cell lines and hepatitis C virus strains are required for the objective evaluation of anti-hepatitis C virus reagents

Ueda, Youki; Mori, Kazuhiko; Ariumi, Yasuo; Ikeda, Masanori; Kato, Naoya

doi:10.1016/j.bbrc.2011.05.061

Cited by 14 publications

(12 citation statements)

References 17 publications

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“…This finding led us to clarify the anti-HCV mechanism of RBV [22], [23]. Furthermore, we demonstrated that plural assay systems including OR6 and ORL8 were required for the objective evaluation of anti-HCV reagents [24]. In that study, we observed that the antimalarial drug artemisinin possessed weak anti-HCV activity, as reported previously [25].…”

Section: Introductionsupporting

confidence: 75%

“…Recently we demonstrated that plural HCV assay systems developed using both HuH-7 and Li23 cell lines or HCV strains belonging to genotype 1b are required for the objective evaluation of anti-HCV candidates [24]. In the present work, we used our previously developed HCV assay systems to evaluate preclinical antimalarial drugs (N-89 and N-251).…”

Section: Resultsmentioning

confidence: 99%

“…RL assay was performed as described previously [20], [24]. Briefly, the cells were plated onto 24-well plates (2×10 4 cells per well) in triplicate and then treated with each reagent at several concentrations for 72 hrs.…”

Section: Methodsmentioning

confidence: 99%

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New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

et al. 2013

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BackgroundPersistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.Methodology/Principal FindingsRecently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.Conclusions/SignificanceWe found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.

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Section: Introductionsupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

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New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

et al. 2013

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“…AB191333)-replicating cell lines, OL (polyclonal; a mixture of approximately 200 clones), OL8 (monoclonal), OL11 (monoclonal), and OL14 (monoclonal) [21], and have been culturing them for more than 4 years. Since we demonstrated that the gene expression profile of Li23 cells was distinct from those in HuH-7 cells [22], and that anti-HCV targets in Li23-derived cells were distinct from those in HuH-7-derived cells [23]–[25], we expected to find distinct HCV variability and diversity from those observed previously in HuH-7-derived cells. To clarify this point, we carried out comprehensive genetic analysis of HCVs obtained from 0-year, 2-year, and 4-year cultures of OL, OL8, OL11, and OL14 cells, and compared them with the original ON/C-5B/QR,KE,SR RNA [21].…”

Section: Introductionmentioning

confidence: 81%

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

et al. 2014

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BackgroundThe most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells.Methodology/Principal FindingsLi23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents.Conclusions/SignificanceLong-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

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“…Ueda et al reported that 6-azauridine showed anti-HCV activity in an HCV drug assay system (ORL8), and the selective index from dividing the CC 50 value by the EC 50 value was 4.1 [48].…”

Section: Discussionmentioning

confidence: 99%

Finding Candidate Drugs for Hepatitis C Based on Chemical-Chemical and Chemical-Protein Interactions

et al. 2014

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Hepatitis C virus (HCV) is an infectious virus that can cause serious illnesses. Only a few drugs have been reported to effectively treat hepatitis C. To have greater diversity in drug choice and better treatment options, it is necessary to develop more drugs to treat the infection. However, it is time-consuming and expensive to discover candidate drugs using experimental methods, and computational methods may complement experimental approaches as a preliminary filtering process. This type of approach was proposed by using known chemical-chemical interactions to extract interactive compounds with three known drug compounds of HCV, and the probabilities of these drug compounds being able to treat hepatitis C were calculated using chemical-protein interactions between the interactive compounds and HCV target genes. Moreover, the randomization test and expectation-maximization (EM) algorithm were both employed to exclude false discoveries. Analysis of the selected compounds, including acyclovir and ganciclovir, indicated that some of these compounds had potential to treat the HCV. Hopefully, this proposed method could provide new insights into the discovery of candidate drugs for the treatment of HCV and other diseases.

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Plural assay systems derived from different cell lines and hepatitis C virus strains are required for the objective evaluation of anti-hepatitis C virus reagents

Cited by 14 publications

References 17 publications

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Finding Candidate Drugs for Hepatitis C Based on Chemical-Chemical and Chemical-Protein Interactions

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