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

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 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 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 Development of a multiplex phenotypic cell-based high throughput screening assay to identify novel hepatitis C virus antivirals

2013 ◽  
Vol 99 (1) ◽  
pp. 6-11 ◽  
Author(s):  
Hee-Young Kim ◽  
Xiaolan Li ◽  
Christopher T. Jones ◽  
Charles M. Rice ◽  
Jean-Michel Garcia ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals Development of a High Throughput Scintillation Proximity Assay for Hepatitis C Virus NS3 Protease That Reduces the Proportion of Competitive Inhibitors Identified

2000 ◽  
Vol 5 (3) ◽  
pp. 153-158 ◽  
Author(s):  
Anne Fowler ◽  
Molly Price-Jones ◽  
Kelvin Hughes ◽  
John Anson ◽  
Russell Lingham ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
Substrate Concentration ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Screening Assay ◽  
Scintillation Proximity Assay ◽  
Competitive Inhibitors ◽  
Ns3 Protease

A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC50 values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.

scholarly journals High-Throughput Screening and Rapid Inhibitor Triage Using an Infectious Chimeric Hepatitis C Virus

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e42609 ◽  
Author(s):  
Michael J. Wichroski ◽  
Jie Fang ◽  
Betsy J. Eggers ◽  
Ronald E. Rose ◽  
Charles E. Mazzucco ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening

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.

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