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BioAssay: AID 602275

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: Set 3

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: Set 3. ..more
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 Tested Compounds
 Tested Compounds
All(78)
 
 
Probe(1)
 
 
Active(63)
 
 
Inactive(16)
 
 
 Tested Substances
 Tested Substances
All(82)
 
 
Probe(1)
 
 
Active(65)
 
 
Inactive(17)
 
 
AID: 602275
Data Source: The Scripps Research Institute Molecular Screening Center (HCV NS3_INH_QFRET_96_IC50_SET 3)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-02-24
Hold-until Date: 2013-02-24
Modify Date: 2013-02-24

Data Table ( Complete ):           View Data Probes    View Active Data    View All Data
BioActive Compounds: Chemical Probe: 1    Active: 63
Related Experiments
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AIDNameTypeComment
1800Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)Screeningdepositor-specified cross reference: Primary screen (HCV NS3 helicase inhibitors in singlicate)
1830Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).Summarydepositor-specified cross reference: Summary (HCV NS3 helicase inhibitors)
1845Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors: biochemical high-throughput screening assay to identify compounds that cause fluorescent intercalator displacement (FID)Screeningdepositor-specified cross reference: Counterscreen (Fluorescent intercalator displacement in singlicate)
1943Fluorescence-based confirmation biochemical high throughput screening assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)Screeningdepositor-specified cross reference: Confirmation screen (HCV NS3 helicase 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.Screeningdepositor-specified cross reference: Counterscreen (Fluorescent intercalator displacement in triplicate)
2172Counterscreen for HCV NS3 helicase inhibitors: Fluorescence-based biochemical high-throughput dose response assay for compounds that cause fluorescent intercalator displacement (FID).Confirmatorydepositor-specified cross reference: Dose response counterscreen (Fluorescent intercalator displacement in triplicate)
2173Fluorescence-based biochemical high throughput dose response assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).Confirmatorydepositor-specified cross reference: Dose response (HCV NS3 helicase 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 NS3Confirmatorydepositor-specified cross reference: Late stage dose response (HCV NS3 helicase inhibitors 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)Confirmatorydepositor-specified cross reference: Late stage dose response counterscreen (Fluorescent intercalator displacement in triplicate )
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 cytotoxicOtherdepositor-specified cross reference: Late stage dose response counterscreen (Cytotoxicity)
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 repliconConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen (Replicon encoded by HCV strains inhibitors)
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 strainsConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen ( encoded by HCV strains inhibitors)
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 2Otherdepositor-specified cross reference: Late stage dose response counterscreen (Helicase encoded by HCV strains inhibitors)
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 cytotoxicConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen (Cytotoxicity)
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 compoundsOthersame project related to Summary assay
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 replicationOthersame project related to Summary assay
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-NS4AConfirmatorysame project related to Summary assay
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 proteinConfirmatorysame project related to Summary assay
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 levelsConfirmatorysame project related to Summary assay
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 DNAConfirmatorysame project related to Summary assay
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 DNAConfirmatorysame project related to Summary assay
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 activityConfirmatorysame 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_IC50_SET 3

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: Set 3.

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, genotype, HCV genotype, 2a(JFH1), 1b(con1), RNA virus, dose response, counterscreen, triplicate, 96, assay provider, inhibitor, inhibition, inhibit, fluorescence, FRET, QFRET, ssDNA oligonucleotide, hairpin, oligonucleotide molecular beacon, quench, quencher, Cy5 fluorophore, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Panel Information
Assays (see Protocol for details)
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1NS3h 1b[con1]6715non-structural polyprotein [Hepatitis C virus] [gi:5441836]
2NS3h 2a[JFH1]6615polyprotein [Recombinant Hepatitis C virus J4/JFH1] [gi:222875993]

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this assay is to verify enzyme activity in a kinetic assay and to examine specificity by examining whether powder samples of purchased or synthesized compounds identified as possible probe candidates inhibit the helicase encoded by the HCV genotypes NS3h_1b[con1] or NS3h_2a[JFH1]. In these assays a ssDNA oligonucleotide molecular beacon substrate 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% of reaction volume of 10 mM ATP into 90% volume reaction mix such that the final 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 1b(con1)) or 5 nM enzyme (NS3h isolation from genotype 2a(JFH1)), 1.0 mM ATP and 5% (v/v) DMSO (final reaction volume 60 uL). 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% tween20 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 Varioskan Flash (Thermo Fisher Scientific, Inc.) or FLUOstar Omega (BMG Labtech, Inc). Cy5 -labeled substrates are measured at excitation wavelength 643 nm (12 nm slit) and emission wavelength 667 nm (12 nm slit) for Varioskan Flash, or excitation wavelength 640 nm (10 nm slit) and emission wavelength 680 (10 nm slit) for FLUOstar.

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 SkanIt Software v 2.4.3 (Thermo Fisher Scientific, 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. 100 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 100 uM.

For each assay, each compound was tested in 1 to 5 independent experiments.

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

Compounds with an IC50 greater than 50 uM were considered inactive. Compounds with an IC50 equal to or less than 50 uM were considered active.

Inactive compounds were scored as 0. Active compounds were scored as follows:

Score = ( 1 - ( ( Ave_IC50 - Min_IC50) / ( ( 100 - Min_IC50 ) / 2 ) ) * 100

Where:

Ave_IC50 is the calculated average IC50 for that compound
Min_IC50 is the minimum Ave_IC50 observed for all compounds in the Assay data set

NS3h 1b[con1] Score: The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 0-0.

NS3h 2a[JFH1] Score: The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.

Overall Outcome and Score:

Compounds that were active in both experiments were considered active, otherwise they were considered inactive.

The PubChem Activity Score is assigned a value of 100 for the probe compound, 50 for active compounds, and 0 for inactive compounds.

The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 0-0.

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)on behalf of the University of Kansas Specialized Chemistry Center. Compounds tested in this assay were purchased and/or synthesized by the University of Kansas Specialized Chemistry Center. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: lead optimization

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: biochemical format: protein format: single protein format

BAO: bioassay specification: assay measurement type: kinetic assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: transcription factor

BAO: meta target: biological process target: viral genome replication

BAO: detection technology: fluorescence: fret: htrf

Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1NS3h_1b(con1): ScoreThe calculated activity score for all Assay 1 experiments1non-structural polyprotein [Hepatitis C virus]Integer
2NS3h_1b(con1): OutcomeHCV Genotype NS3h_1b[con1]-assay outcome, one of active, inactive, or inconclusive.1Outcome
3NS3h_1b(con1): Average IC50*Average IC50 value of the IC50s of experiments 4 and 5 and experiments 1-3 where qualifier is "=".1FloatμM
4NS3h_1b(con1): Qualifier [Exp. 1]Experiment 1: Activity 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
5NS3h_1b(con1): IC50 [Exp. 1]Experiment 1: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.1FloatμM
6NS3h_1b(con1): Qualifier [Exp. 2]Experiment 2: Activity 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
7NS3h_1b(con1): IC50 [Exp. 2]Experiment 2: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 2.1FloatμM
8NS3h_1b(con1): Qualifier [Exp. 3]Experiment 3: Activity 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
9NS3h_1b(con1): IC50 [Exp. 3]Experiment 3: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 3.1FloatμM
10NS3h_1b(con1): IC50 [Exp. 4]Experiment 4: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 4.1FloatμM
11NS3h_1b(con1): IC50 [Exp. 5]Experiment 5: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 5.1FloatμM
12NS3h_2a(JFH1): ScoreThe calculated activity score for all Assay 2 experiments2polyprotein [Recombinant Hepatitis C virus J4/JFH1]Integer
13NS3h_2a(JFH1): OutcomeHCV Genotype NS3h_2a[JFH1]-assay outcome, one of active, inactive, or inconclusive.2Outcome
14NS3h_2a(JFH1): Average IC50*Average IC50 value of the IC50s of experiments 4 and 5 and experiments 1-3 where qualifier is "=".2FloatμM
15NS3h_2a(JFH1): Qualifier [Exp. 1]Experiment 1: Activity 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
16NS3h_2a(JFH1): IC50 [Exp. 1]Experiment 1: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.2FloatμM
17NS3h_2a(JFH1): Qualifier [Exp. 2]Experiment 2: Activity 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
18NS3h_2a(JFH1): IC50 [Exp. 2]Experiment 2: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 2.2FloatμM
19NS3h_2a(JFH1): Qualifier [Exp. 3]Experiment 3: Activity 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
20NS3h_2a(JFH1): IC50 [Exp. 3]Experiment 3: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 3.2FloatμM
21NS3h_2a(JFH1): IC50 [Exp. 4]Experiment 4: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 4.2FloatμM
22NS3h_2a(JFH1): IC50 [Exp. 5]Experiment 5: The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar, or NT if the compound was not tested in Experiment 5.2FloatμM

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

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