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BioAssay: AID 623966

Late 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 protein

Name: Late 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 protein. ..more
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 Tested Compounds
 Tested Compounds
All(49)
 
 
Active(37)
 
 
Inactive(12)
 
 
 Tested Substances
 Tested Substances
All(50)
 
 
Active(38)
 
 
Inactive(12)
 
 
 Related BioAssays
 Related BioAssays
AID: 623966
Data Source: The Scripps Research Institute Molecular Screening Center (HCV NS3_INH_FLUOPOL_384_1X%DIS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-04-02
Hold-until Date: 2013-03-30
Modify Date: 2013-03-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 37
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 (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).Summary depositor-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)Screening depositor-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)Screening depositor-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.Screening depositor-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).Confirmatory depositor-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).Confirmatory depositor-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 NS3Confirmatory depositor-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)Confirmatory depositor-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 cytotoxicOther depositor-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 repliconConfirmatory depositor-specified cross reference: Late stage dose response (Replication of HCV RNA replicon 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 strainsConfirmatory depositor-specified cross reference: Late stage dose response counterscreen (Helicase 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 2Other depositor-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 cytotoxicConfirmatory depositor-specified cross reference: Late stage dose response counterscreen (Cytotoxicity)
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 same project related to Summary assay
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 same 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 replicationOther same 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-NS4AConfirmatory same 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 levelsConfirmatory same 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 DNAConfirmatory same 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 DNAConfirmatory same 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 activityConfirmatory 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_FLUOPOL_384_1X%DIS

Name: Late 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 protein.

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, 384, assay provider, inhibitor, inhibition, inhibit, E. coli single stranded DNA binding protein, SSB, 5' Cy5, Cy5-dT15), fluorescence polarization, 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 biochemical assay is to determine the ability of compounds to displace the E. coli single stranded DNA binding protein (SSB) from its substrate oligonucleotide. In this biochemical assay, the fluorescence polarization of a single strand of DNA with a 5' Cy5 fluorophore (Cy5-dT15) incubated with E. coli SSB and test compound is monitored. Fluorescence polarization increases when the E. coli SSB binds the substrate. As designed, compounds that interfere with binding decrease the fluorescence polarization.

Protocol Summary:

Assays were performed in 60 uL in 384-well black microplates. All assays contained 5 nM Cy5-dT15 DNA (5'- Cy5 TT TTT TTT TTT TTT T -3'), 20 nM SSB, 25 mM MOPS, pH 7.5, 1.25 mM MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) Tween20, and 0.025 mM DTT. Compounds dissolved in DMSO were added and Cy5 fluorescence polarization was read using a G-factor calculated from a well lacking SSB or test compounds. Percent binding was calculated according to the equation below.

%_Binding = ( 1 - ( ( Fc - F[+] / F[-] - F[+] ) / ( F[-] - F[+] ) ) * 100

Where:

Fc is the fluorescence polarization in the presence of the compound
F[-] is the average no enzyme negative controls
F[+] is the average positive controls (200 nM dT20).

Percent displacement was calculated by subtracting the percent binding from 100.

IC50 values were calculated from a normalized dose response curve using GraphPad Prism (v. 5).

PubChem Activity Outcome and Score:

Compounds with an IC50 of 50 uM or less are considered active, compounds with an IC50 of greater than 50 uM are considered inactive.

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 active compounds is 100-26, and for inactive compounds 26-0.

List of Reagents:

E. coli SSB (New England Biolabs, part M301A)
Cy5-dT15 (Integrated DNA Technologies Inc, custom synthesized)
dT20 (Integrated DNA Technologies Inc, custom synthesized)
MOPS (Fisher Scientific, part BP308-100)
Magnesium Chloride (Fisher Scientific, part BP214-500)
384-well plates (Corning Costar, black, part 3573)
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 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
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: alternate confirmatory

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: biochemical format: protein format: protein complex format

BAO: bioassay specification: assay measurement type: endpoint 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: enzyme regulator

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

BAO: detection technology: fluorescence: fluorescence polarization

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identified 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.String
2IC50*The concentration at which 50 percent of the activity in the antagonist assay is observed (IC50); shown in micromolarFloatμM
3Standard DeviationStandard deviation of the IC50.Float
4Displacement at 100 uM (100μM**)Value of percent displaced at 100 uM compound concentrationFloat%
5Displacement at 67 uM (67μM**)Value of percent displaced at 67 uM compound concentrationFloat%
6Displacement at 44.89 uM (44.89μM**)Value of percent displaced at 44.89 uM compound concentrationFloat%
7Displacement at 30.08 uM (30.08μM**)Value of percent displaced at 30.08 uM compound concentrationFloat%
8Displacement at 20.15 uM (20.15μM**)Value of percent displaced at 20.15 uM compound concentrationFloat%
9Displacement at 13.5 uM (13.5μM**)Value of percent displaced at 13.5 uM compound concentrationFloat%
10Displacement at 9.05 uM (9.05μM**)Value of percent displaced at 9.05 uM compound concentrationFloat%
11Displacement at 6.06 uM (6.06μM**)Value of percent displaced at 6.06 uM compound concentrationFloat%
12Displacement at 4.06 uM (4.06μM**)Value of percent displaced at 4.06 uM compound concentrationFloat%
13Displacement at 2.72 uM (2.72μM**)Value of percent displaced at 2.72 uM compound concentrationFloat%
14Displacement at 1.82 uM (1.82μM**)Value of percent displaced at 1.82 uM compound concentrationFloat%
15Displacement at 1.22 uM (1.22μM**)Value of percent displaced at 1.22 uM compound concentrationFloat%
16Displacement at 0.82 uM (0.82μM**)Value of percent displaced at 0.82 uM compound concentrationFloat%
17Displacement at 0.55 uM (0.55μM**)Value of percent displaced at 0.55 uM compound concentrationFloat%
18Displacement at 0.37 uM (0.37μM**)Value of percent displaced at 0.37 uM compound concentrationFloat%
19Displacement at 0.25 uM (0.25μM**)Value of percent displaced at 0.25 uM compound concentrationFloat%

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

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