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BioAssay: AID 623970

Late 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 DNA

Name: Late 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 DNA. ..more
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 Tested Compounds
 Tested Compounds
All(68)
 
 
Active(34)
 
 
Inactive(35)
 
 
 Tested Substances
 Tested Substances
All(71)
 
 
Active(35)
 
 
Inactive(36)
 
 
 Related BioAssays
 Related BioAssays
AID: 623970
Data Source: The Scripps Research Institute Molecular Screening Center (HCV NS3_INH_FLUO_96_%DISPLACEMENT_SYBR)
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 ):           Active    All
BioActive Compounds: 34
Depositor Specified Assays
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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)screeningPrimary 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).summarySummary (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)screeningCounterscreen (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)screeningConfirmation 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.screeningCounterscreen (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).confirmatoryDose 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).confirmatoryDose 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 NS3confirmatoryLate 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)confirmatoryLate 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 cytotoxicLate 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 repliconconfirmatoryLate 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 strainsconfirmatoryLate 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 cytotoxicconfirmatoryLate stage dose response counterscreen (Cytotoxicity)
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 2otherLate stage dose response counterscreen (Helicase encoded by HCV strains inhibitors)
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_FLUO_96_%DISPLACEMENT_SYBR

Name: Late 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 DNA.

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, DNA hairpin oligonucleotide, ethidium bromide, intercalating, 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 is to determine dose response curves for powder samples of compounds identified as potential probe candidates. This assay serves to determine whether the compounds are nonselective due to direct DNA binding. In this biochemical assay, a DNA hairpin oligonucleotide is incubated with test compound and a fluorescent intercalating agent, SYBR Green I. Upon binding to the DNA molecule, the fluorescence of the intercalating agent increases. As designed, test compounds that bind to the DNA molecule will compete with and displace the intercalating agent, resulting in a decrease in well fluorescence.

Protocol Summary:

Compounds were tested in triplicate in an 8-point 1:2 dilution series starting at a nominal test concentration of 100 uM. Each 60 uL reaction contained 25 mM MOPS pH 6.5, 0.64 uM MBHA DNA substrate, 1x SYBR Green I, and various concentrations of test compound. Each reaction was incubated for 10 minutes at 25 Celsius before fluorescence of SYBR Green I was monitored using excitation and emission wavelengths of 497 and 527 nm, respectively, on a VarioSkan (Thermo Scientific) plate reader in white 96-well half-area plates. The amount of SYBR Green I-DNA complex fluorescence was used to estimate the ability of compounds to bind DNA, and therefore displace the fluorescent intercalator (SYBR Green I). Percent binding was calculated as follows.

%_Displacement = ( 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 (100 uM berenil)

An IC50 value was calculated from a normalized dose response curve for all assays using GraphPad Prism (v. 5).

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than or equal to 100 uM are inactive. Compounds with an IC50 less than 100 uM are 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 active compounds is 100-1, and for inactive compounds 0-0.

List of Reagents:

DNA hairpin oligonucleotide (Integrated DNA Technologies Inc, custom synthesized)
Intercalating agent (SYBR Green I) (Life Technologies, S-7585)
MOPS (Fisher Scientific, part BP308-100)
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 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 intensity

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
2Average IC50*The concentration at which 50 percent of the activity is observed (IC50); shown in micromolarFloatμM
3Standard ErrorStandard Error of IC50 calculationFloat
4Displacement at 100 uM [1] (100μM**)Value of percent displacement at 100 uM compound concentrationFloat%
5Displacement at 50 uM [1] (50μM**)Value of percent displacement at 50 uM compound concentrationFloat%
6Displacement at 25 uM [1] (25μM**)Value of percent displacement at 25 uM compound concentrationFloat%
7Displacement at 12.5 uM [1] (12.5μM**)Value of percent displacement at 12.5 uM compound concentrationFloat%
8Displacement at 6.25 uM [1] (6.25μM**)Value of percent displacement at 6.25 uM compound concentrationFloat%
9Displacement at 3.13 uM [1] (3.13μM**)Value of percent displacement at 3.13 uM compound concentrationFloat%
10Displacement at 1.56 uM [1] (1.56μM**)Value of percent displacement at 1.56 uM compound concentrationFloat%
11Displacement at 0.78 uM [1] (0.78μM**)Value of percent displacement at 0.78 uM compound concentrationFloat%
12Displacement at 100 uM [2] (100μM**)Value of percent displacement at 100 uM compound concentrationFloat%
13Displacement at 50 uM [2] (50μM**)Value of percent displacement at 50 uM compound concentrationFloat%
14Displacement at 25 uM [2] (25μM**)Value of percent displacement at 25 uM compound concentrationFloat%
15Displacement at 12.5 uM [2] (12.5μM**)Value of percent displacement at 12.5 uM compound concentrationFloat%
16Displacement at 6.25 uM [2] (6.25μM**)Value of percent displacement at 6.25 uM compound concentrationFloat%
17Displacement at 3.13 uM [2] (3.13μM**)Value of percent displacement at 3.13 uM compound concentrationFloat%
18Displacement at 1.56 uM [2] (1.56μM**)Value of percent displacement at 1.56 uM compound concentrationFloat%
19Displacement at 0.78 uM [2] (0.78μM**)Value of percent displacement at 0.78 uM compound concentrationFloat%
20Displacement at 100 uM [3] (100μM**)Value of percent displacement at 100 uM compound concentrationFloat%
21Displacement at 50 uM [3] (50μM**)Value of percent displacement at 50 uM compound concentrationFloat%
22Displacement at 25 uM [3] (25μM**)Value of percent displacement at 25 uM compound concentrationFloat%
23Displacement at 12.5 uM [3] (12.5μM**)Value of percent displacement at 12.5 uM compound concentrationFloat%
24Displacement at 6.25 uM [3] (6.25μM**)Value of percent displacement at 6.25 uM compound concentrationFloat%
25Displacement at 3.13 uM [3] (3.13μM**)Value of percent displacement at 3.13 uM compound concentrationFloat%
26Displacement at 1.56 uM [3] (1.56μM**)Value of percent displacement at 1.56 uM compound concentrationFloat%
27Displacement at 0.78 uM [3] (0.78μM**)Value of percent displacement at 0.78 uM compound concentrationFloat%
28Displacement at 100 uM [4] (100μM**)Value of percent displacement at 100 uM compound concentrationFloat%
29Displacement at 50 uM [4] (50μM**)Value of percent displacement at 50 uM compound concentrationFloat%
30Displacement at 25 uM [4] (25μM**)Value of percent displacement at 25 uM compound concentrationFloat%
31Displacement at 12.5 uM [4] (12.5μM**)Value of percent displacement at 12.5 uM compound concentrationFloat%
32Displacement at 6.25 uM [4] (6.25μM**)Value of percent displacement at 6.25 uM compound concentrationFloat%
33Displacement at 3.13 uM [4] (3.13μM**)Value of percent displacement at 3.13 uM compound concentrationFloat%
34Displacement at 1.56 uM [4] (1.56μM**)Value of percent displacement at 1.56 uM compound concentrationFloat%
35Displacement at 0.78 uM [4] (0.78μM**)Value of percent displacement at 0.78 uM compound concentrationFloat%

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

Data Table (Concise)
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