scholarly journals Novel Chimeric Genotype 1b/2a Hepatitis C Virus Suitable for High-Throughput Screening

2007 ◽  
Vol 52 (2) ◽  
pp. 666-674 ◽  
Author(s):  
Yingjia Zhang ◽  
Peter Weady ◽  
Rohit Duggal ◽  
Weidong Hao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening ◽  
Luciferase Reporter ◽  
Structural Protein ◽  
Genotype 1 ◽  
Luciferase Reporter Gene ◽  
Subgenomic Replicon ◽  
Viral Particles

ABSTRACT A major obstacle in hepatitis C virus (HCV) research has been the lack of a permissive cell culture system that produces infectious viral particles. Significant breakthroughs have been achieved lately in establishing such culture systems. Yet to date, there are no reports of the applications of any of these systems in HCV drug screening. Here, we report the generation of two monocistronic, chimeric genotype 1 full-length HCV genome molecules. These molecules, C33J-Y835C-UBI and C33J-Y835C-FMDV2A, both contain the structural protein region from genotype 1 (subtype 1b, Con1) and the remaining region from the genotype 2a (JFH1) clone. Both contain the humanized Renilla luciferase reporter gene which is separated from the rest of the HCV open reading frame by two different cleavage sites. The viral RNAs replicated efficiently in transfected cells. Viral particles produced were infectious in naïve Huh7.5 cells, and the infectivity could be blocked by monoclonal antibody against a putative HCV entry cofactor, CD81. A pilot high-throughput screen of 900 unknown compounds was executed by both the genotype 2a subgenomic replicon system and the infectious system. Thirty-one compounds were identified as hits by both systems, whereas 78 compounds were identified as hits only for the infectious system, suggesting that the infectious system is capable of identifying inhibitors targeting the viral structural proteins and steps involving them in the viral life cycle. The infectious HCV system developed here provides a useful and versatile tool which should greatly facilitate the identification of HCV inhibitors currently not identified by the subgenomic replicon system.

scholarly journals Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

2009 ◽  
Vol 53 (11) ◽  
pp. 4825-4834 ◽  
Author(s):  
Kao-Lu Pan ◽  
Jin-Ching Lee ◽  
Hsing-Wen Sung ◽  
Teng-Yuang Chang ◽  
John T.-A. Hsu
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter System ◽  
Viral Protease ◽  
Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

scholarly journals In VitroActivity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor

2014 ◽  
Vol 59 (3) ◽  
pp. 1505-1511 ◽  
Author(s):  
Warren Kati ◽  
Gennadiy Koev ◽  
Michelle Irvin ◽  
Jill Beyer ◽  
Yaya Liu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clinical Isolates ◽  
Genotype 1 ◽  
Subgenomic Replicon ◽  
Hcv Genotype ◽  
Genotype 1A ◽  
Treatment Naïve ◽  
Hcv Genotype 1 ◽  
Hcv Ns5b

ABSTRACTDasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

scholarly journals Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

2007 ◽  
Vol 73 (20) ◽  
pp. 6436-6443 ◽  
Author(s):  
Andreas Urban ◽  
Stefan Eckermann ◽  
Beate Fast ◽  
Susanne Metzger ◽  
Matthias Gehling ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Biosynthesis ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Environmental Biology ◽  
Drug Candidates ◽  
Novel Drug

ABSTRACT Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

scholarly journals Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

2013 ◽  
Vol 58 (2) ◽  
pp. 995-1004 ◽  
Author(s):  
Zongyi Hu ◽  
Keng-Hsin Lan ◽  
Shanshan He ◽  
Manju Swaroop ◽  
Xin Hu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dose Range ◽  
Hcv Infection ◽  
Replication Cycle ◽  
Screening Methods ◽  
Functional Features ◽  
Pharmacologically Active

ABSTRACTTherapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Novel Alpha Interferon (IFN-α) Variant with Improved Inhibitory Activity against Hepatitis C Virus Genotype 1 Replication Compared to IFN-α2b Therapy in a Subgenomic Replicon System

2006 ◽  
Vol 50 (12) ◽  
pp. 3984-3991 ◽  
Author(s):  
Vanessa Escuret ◽  
Amaury Martin ◽  
David Durantel ◽  
Romain Parent ◽  
Olivier Hantz ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Inhibitory Activity ◽  
Alpha Interferon ◽  
Genotype 1 ◽  
Subgenomic Replicon ◽  
Hcv Genotype ◽  
Long Term Treatment ◽  
Hcv Genotype 1 ◽  
In Cells

ABSTRACT Hepatitis C virus (HCV) treatment is based on the association of pegylated alpha interferon (IFN-α) and ribavirin. To improve the level of sustained virological response to treatment, especially in patients infected with HCV genotype 1, new IFNs with improved efficacy and toxicity profiles may be developed. In this report, we show that, in the BM4-5 cell line harboring an HCV subgenomic replicon, a novel and naturally occurring human IFN-α17 variant, GEA007.1, which was discovered by using an original population genetics-based drug discovery approach, inhibits HCV genotype 1 RNA replication more efficiently than does IFN-α2b. Moreover, we show that complete viral clearance is obtained in BM4-5 cells after long-term treatment with GEA007.1, while HCV subgenomic RNA is still detected in cells treated with other IFN-α variants or with standard IFN-α2b. Eventually, we demonstrate that the better inhibitory activity of GEA007.1 compared to that of standard IFN-α is likely to be due to stronger and faster activation of the JAK-STAT signaling pathway and to broader expression of IFN-α-responsive genes in cells. Our results demonstrate a superior inhibitory activity of GEA007.1 over that of IFN-α2b in the HCV replicon system. Clinical trials are required to determine whether GEA007.1 could be a potent “next generation” IFN for the treatment of HCV infection, especially in nonresponders or relapsing patients infected with HCV genotype 1 who currently represent a clinical unmet need.

scholarly journals A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase

2013 ◽  
Vol 18 (9) ◽  
pp. 1027-1034 ◽  
Author(s):  
Auda A. Eltahla ◽  
Kurt Lackovic ◽  
Christopher Marquis ◽  
John-Sebastian Eden ◽  
Peter A. White
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Specific Activity ◽  
Hcv Rna ◽  
High Throughput Screen ◽  
Rdrp Activity

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 “lead-like” compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC50 of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

scholarly journals High-Throughput Screening, Discovery, and Optimization To Develop a Benzofuran Class of Hepatitis C Virus Inhibitors

2015 ◽  
Vol 17 (10) ◽  
pp. 641-652 ◽  
Author(s):  
Shanshan He ◽  
Prashi Jain ◽  
Billy Lin ◽  
Marc Ferrer ◽  
Zongyi Hu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening
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