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BioAssay: AID 485301

Late 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

Name: Late 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. ..more
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 Tested Compounds
 Tested Compounds
All(9)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Inactive(16)
 
 
AID: 485301
Data Source: The Scripps Research Institute Molecular Screening Center (HCV NS3_INH_QFRET_96_1XIC50)
BioAssay Type: Panel, Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-09-29
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)
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 depositor-specified cross reference
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
588360Late-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 cytotoxicConfirmatory 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: HCV NS3_INH_QFRET_96_1XIC50

Name: Late 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.

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, HCV, NS3, NS3 helicase, hepatitis, RNA virus, dose response, counterscreen, triplicate, 96, assay provider, inhibitor, inhibition, inhibit, fluorescence, FRET, QFRET, hairpin, oligonucleotide molecular beacon, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Panel Information
Assays
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1NS3h 2a (JFH1)8polyprotein [Recombinant Hepatitis C virus J4/JFH1] [gi:222875993]
A truncated NS3 protein lacking a protease domain (NS3 residues 166-631) of HCV genotype 2a (isolate JFH1).
2NS3h 1b (J4)16polyprotein [Hepatitis C virus subtype 1b] [gi:3098633]
A truncated NS3 protein lacking a protease domain (NS3 residues 166-631) of HCV genotype 1b (isolate J4).
3NS3h 1b (con1)16non-structural polyprotein [Hepatitis C virus] [gi:5441836]
A truncated NS3 protein lacking a protease domain (NS3 residues 166-631) of HCV genotype 1b (isolate con1).

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to verify activity in a kinetic assay, and to examine specificity by examining whether purchased or synthesized compounds identified as possible probe candidates inhibit the helicase encoded by a variety of diverse HCV genotypes. In this assay, a ssDNA oligonucleotide molecular beacon substrate (NS3h_2a[JFH1], NS3h_1b[J4], or NS3h_1b[con1]), featuring a 5' fluorescent Cy5 moiety and a 3' quencher, is annealed to a second longer DNA oligonucleotide. Upon strand separation by NS3 helicase and ATP, the beacon strand forms an intramolecular hairpin that brings the tethered fluorophore and quencher molecules into juxtaposition, quenching fluorescence. As designed, compounds that inhibit helicase activity will prevent hairpin formation and interaction of the Cy5 fluorophore and quencher, thus preventing quenching of well fluorescence.
Protocol Summary:
Assays are initiated by rapidly mixing 10 uL of 10 mM ATP into a 90 uL reaction mix such that the final 100 uL reaction contained 25 mM MOPS pH 6.5, 1.25 mM MgCl2, 5 nM [Cy5-labeled]substrate, 12.5 nM enzyme (NS3h isolation from genotype 2a[JFH1], 1b[con1], or 1b[J4]), 1.0 mM ATP and 5% (v/v) DMSO. Compounds are diluted in DMSO at 20X concentration and added as 5% of the reaction mixture; control (no inhibitor) reactions include DMSO only. Enzyme is diluted in buffer containing 25 mM MOPS pH 6.5, 1 mM DTT, 0.1 mg/mL BSA and 0.2% Tween 20 to 20X final concentration and comprises 5% (v/v) of the reaction mixture. Reactions are performed at 23 C in low volume white 96-well microplates and monitored with a Varian Cary Eclipse fluorescence spectrophotometer. Cy5 -labeled substrates are measured at excitation wavelength 643 nm (5 nm slit) and emission wavelength 667 nm (10 nm slit).
Initial rates of fluorescence decrease after ATP addition were plotted versus compound concentration to calculate IC50 values. For each test compound, reaction velocity (relative fluorescence units [RFU]/min) was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted using Assay Prism Version 5 (Graphpad Sofware, Inc.). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 500 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 500 uM.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 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.
NS3h 2a (JFH1) Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
NS3h 1b (J4) Score: The PubChem Activity Score range for active compounds is 0-0. There are no active compounds.
NS3h 1b (con1) Score: The PubChem Activity Score range for active compounds is 0-0. There are no active compounds.
Overall Outcome and Score:
The overall outcome was active if the compound was active in at least one panel, inactive otherwise.
The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
NS3 helicase fragment (supplied by Assay Provider)
Cy5/quencher-labeled molecular beacon (Integrated DNA Technologies Inc, custom synthesized)
Thioflavine S (Sigma-Aldrich, part T1892)
MOPS (Fisher-Biotech, part BP308-100)
ATP (Fisher-BioReagents, part BP413-25)
Magnesium Chloride (Fisher-Biotech, part BP214-500)
Assay Buffer (supplied by Assay Provider)
96-well plates (Corning Costar, white half volume, part 3693)
Comment
This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compounds tested in this assay were purchased and/or synthesized 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: Biochemical
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1NS3h_2a (JFH1): OutcomeHCV Genotype NS3h 2a(JFH1)assay outcome, one of active, inactive, or not tested.1polyprotein [Recombinant Hepatitis C virus J4/JFH1]Outcome
2NS3h_2a (JFH1): ScoreThe BioAssay activity ranking score1Integer
3NS3h_2a (JFH1): QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.1String
4NS3h_2a (JFH1): IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.1FloatμM
5NS3h_1b (J4): OutcomeHCV Genotype NS3h 1b(J4) --assay outcome, one of active, inactive, or not tested.2polyprotein [Hepatitis C virus subtype 1b]Outcome
6NS3h_1b (J4): ScoreThe BioAssay activity ranking score2Integer
7NS3h_1b (J4): QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.2String
8NS3h_1b (J4): IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.2FloatμM
9NS3h_1b (con1): OutcomeHCV Genotype NS3h 1b(con1) --assay outcome, one of active, inactive, or not tested.3non-structural polyprotein [Hepatitis C virus]Outcome
10NS3h_1b (con1): ScoreThe BioAssay activity ranking score3Integer
11NS3h_1b (con1): QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.3String
12NS3h_1b (con1): IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.3FloatμM

* Activity Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R03 MH085690-01

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