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BioAssay: AID 588360

Late-stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds that inhibit replication of HCV RNA replicon are cytotoxic

Name: Late-stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds that inhibit replication of HCV RNA replicon are cytotoxic. ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Inactive(8)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Inactive(8)
 
 
 Related BioAssays
 Related BioAssays
AID: 588360
Data Source: The Scripps Research Institute Molecular Screening Center (AVA5CYTOX_INH_96_CC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-09-15
Hold-until Date: 2011-09-16
Modify Date: 2011-09-16

Data Table ( Complete ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
1800Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)Screening depositor-specified cross reference: Primary screen (NS3 inhibitors in singlicate)
1830Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).Summary depositor-specified cross reference: Summary (NS3 inhibitors)
1845Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors: biochemical high-throughput screening assay to identify compounds that cause fluorescent intercalator displacement (FID)Screening depositor-specified cross reference: Counterscreen (FID in singlicate)
1943Fluorescence-based confirmation biochemical high throughput screening assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)Screening depositor-specified cross reference: Confirmation screen (NS3 inhibitors in triplicate)
1945Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors: biochemical high-throughput screening assay to identify compounds that cause fluorescent intercalator displacement (FID) in triplicate.Screening depositor-specified cross reference: Counterscreen (FID in triplicate)
2172Counterscreen for HCV NS3 helicase inhibitors: Fluorescence-based biochemical high-throughput dose response assay for compounds that cause fluorescent intercalator displacement (FID).Confirmatory depositor-specified cross reference: Dose response counterscreen (FID in triplicate)
2173Fluorescence-based biochemical high throughput dose response assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).Confirmatory depositor-specified cross reference: Dose response (NS3 inhibitors in triplicate)
2474Late stage results for the probe development effort to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for inhibitors of NS3Confirmatory depositor-specified cross reference: Dose response counterscreen (ATP hydrolysis in triplicate)
2476Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for compounds that cause fluorescent intercalator displacement (FID)Confirmatory depositor-specified cross reference: Dose response counterscreen (FID in triplicate)
602275Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 3Other1 depositor-specified cross reference
623961Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based assay to determine cytotoxicity of compoundsOther depositor-specified cross reference
623962Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based assay to determine whether compounds inhibit HCV replicationOther depositor-specified cross reference
623964Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence-based biochemical assay to determine whether compounds inhibit the HCV protease NS3-NS4AConfirmatory depositor-specified cross reference
623966Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence polarization-based biochemical assay to determine whether compounds can displace the E. coli single stranded DNA binding proteinConfirmatory depositor-specified cross reference
623968Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): RT-PCR-based cell-based assay to determine the effect of compounds on RNA levelsConfirmatory depositor-specified cross reference
623970Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence-based biochemical assay with SYBR to determine whether compounds bind DNAConfirmatory depositor-specified cross reference
623972Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence-based biochemical assay with EtBr to determine whether compounds bind DNAConfirmatory depositor-specified cross reference
623973Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): absorbance-based biochemical assay to determine whether compounds inhibit ATPase activityConfirmatory depositor-specified cross reference
463231Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds that inhibit replication of HCV RNA replicon are cytotoxicOther same project related to Summary assay
463235Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds inhibit replication of HCV RNA repliconConfirmatory same project related to Summary assay
485301Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strainsConfirmatory same project related to Summary assay
504419Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 2Other same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frick, New York Medical College
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH085690-01
Grant Proposal PI: David Frick, New York Medical College
External Assay ID: AVA5CYTOX_INH_96_CC50

Name: Late-stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds that inhibit replication of HCV RNA replicon are cytotoxic.

Description:

The flavivirus Hepatitis C Virus (HCV) is a major cause of liver failure and hepatocellular cancer, with about 170 million people infected worldwide (1). The HCV has a small RNA genome that is directly translated by the infected host cell into a single precursor polyprotein that is processed by enzymatic cleavage into 10 proteins of diverse function. The non-structural proteins include p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, and are responsible for the replication and packaging of the HCV genome into capsids formed by the structural proteins (core, E1, E2)(2). Replication of HCV in human cells requires the action of the HCV non-structural protein 3 (NS3). This enzyme exhibits dual NTPase/helicase activities and functions to unwind DNA/DNA, RNA/RNA, and RNA/DNA duplexes by disrupting hydrogen bonds that hold the two strands together (3). The HCV NS3 helicase mediates the active form of duplex unwinding, and thus is dependent upon NTP and at least two nucleic acid binding sites on the NS3 surface (3). HCV NS3 is able to target homotypic and heterotypic duplexes because the interaction between the enzyme and the DNA or RNA substrate is mediated by phosphate groups and not by the nucleotide base or sugar moieties (4). The current absence of a vaccine to prevent HCV infection (5), along with knockout studies showing that the helicase and/or NTPase activities are essential for viral replication (6), and the lack of HCV genotype-specific differences in helicase residues and activities (7), support a role for NS3 as an important pathogenic component of HCV. The identification of specific inhibitors of HCV NS3 helicase will add insights into the biology of HCV infection and replication, and serve as valuable tools for inhibiting HCV replication in human cells.

References:

1. Hoofnagle, J.H., Course and outcome of hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. s21-s29.
2. Frick, D.N., The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol, 2007. 9(1): p. 1-20.
3. Borowski, P., Schalinski, S., and Schmitz, H., Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy. Antiviral Res, 2002. 55(3): p. 397-412.
4. Kim, J.L., Morgenstern, K.A., Griffith, J.P., Dwyer, M.D., Thomson, J.A., Murcko, M.A., Lin, C., and Caron, P.R., Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure, 1998. 6(1): p. 89-100.
5. Yang, J.P., Zhou, D., and Wong-Staal, F., Screening of small-molecule compounds as inhibitors of HCV entry. Methods Mol Biol, 2009. 510: p. 295-304.
6. Gu, B., Liu, C., Lin-Goerke, J., Maley, D.R., Gutshall, L.L., Feltenberger, C.A., and Del Vecchio, A.M., The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J Virol, 2000. 74(4): p. 1794-800.
7. Cho, H.S., Ha, N.C., Kang, L.W., Chung, K.M., Back, S.H., Jang, S.K., and Oh, B.H., Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem, 1998. 273(24): p. 15045-52.

Keywords:

late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, NIAID, Antimicrobial Acquisition and Coordinating Facility, AACF, HCV, NS3, NS3 helicase, hepatitis, RNA virus, counterscreen, dose response, inhibitor, inhibition, inhibit, cytotoxicity, neutral red dye, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:
The purpose of this assay, performed by NIAIDs Antimicrobial Acquisition and Coordinating Facility (AACF) (http://niaid-aacf.org/protocols/HCV.htm) is to determine cytotoxicity of samples of purchased compounds identified as possible probe candidates. In this assay, cytotoxicity is assessed by neutral red dye uptake. As designed, compounds that are cytotoxic will kill cells and decrease the amount of red dye taken up.
Protocol Summary:
Cytotoxicity is assessed in an assay using the stably-expressing HCV replicon cell line, AVA5 (sub-genomic [CON1], genotype 1b) maintained as sub-confluent cultures on 96-well plates. Cytotoxicity is assessed by neutral red dye uptake.
CC50 values are calculated by linear regression analysis (MS EXCEL(R), QuattroPro(R)) using data combined from all treated cultures. CC50 is the drug concentration at which a 2-fold lower level of neutral red dye uptake (relative to the average levels in untreated cultures) is observed.
PubChem Activity Outcome and Score:
Compounds with an CC50 greater than 10 uM were considered inactive. Compounds with an CC50 equal to or less than 10 uM were considered active.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
Reagents were provided by the AACF program.
Comment
This assay was performed by the NIAIDs Antimicrobial Acquisition and Coordinating Facility (AACF), and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compounds tested in this assay were purchased by the University of Kansas Specialized Chemistry Center. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDS listed in the Related Bioassays section of this AID.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data CC50 came from a fitted curve or was determined manually to be less than or greater than its listed CC50 concentration.String
2CC50*The concentration at which 50 percent of surviving cells are observed; (CC50) shown in micromolar.FloatμM

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH085690-01

Data Table (Concise)
Data Table ( Complete ):     View All Data
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