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BioAssay: AID 1943

Fluorescence-based confirmation biochemical high throughput screening assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)

Name: Fluorescence-based confirmation biochemical high throughput screening assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3). ..more
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 Tested Compounds
 Tested Compounds
All(1597)
 
 
Active(500)
 
 
Inactive(1097)
 
 
 Tested Substances
 Tested Substances
All(1598)
 
 
Active(500)
 
 
Inactive(1098)
 
 
AID: 1943
Data Source: The Scripps Research Institute Molecular Screening Center (NS3HCVDNA_INH_FLINT_1536_3X%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-09-09
Modify Date: 2010-06-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 500
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1830Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).summary Summary AID.
1800Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)screening Primary screening assay.
2173Fluorescence-based biochemical high throughput dose response assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).confirmatory
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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: NS3HCVDNA_INH_FLINT_1536_3X%INH

Name: Fluorescence-based confirmation biochemical high throughput screening assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).

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:

HCV, NS3, NS3 helicase, hepatitis, RNA virus, confirmation, HTS, high throughput screen, 1536, inhibitor, FLINT, fluorescence, fluorescence intensity, FLINT, 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 confirm activity of compounds identified as active in s set of previous experiments entitled, "Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)" (AID 1800). In this assay, 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 at 60 minutes (T60) will prevent hairpin formation and interaction of the Cy5 fluorophore and quencher, thus preventing quenching of well fluorescence. A T0 fluorescence measurement is also performed to identify compounds that quench and enhance Cy5 fluorescence. Compounds were tested in triplicate at a final nominal concentration of 10.9 micromolar.

Protocol Summary:
Prior to the start of the assay, 4 microliters of Assay Buffer (25 mM MOPS pH 6.5, 1.25 mM MgCl2, 0.1 mM DTT, 12.5 mM Tween20, 6 micrograms/mL BSA) containing 13.89 nM NS3 helicase fragment and 5.56 nM NS3 substrate were dispensed into wells of a 1536 microtiter plate. Next, 55 nL of test compound in DMSO, thioflavine S (110 micromolar final concentration), or DMSO alone (0.8% final concentration) were added to the appropriate wells.

The assay was started by dispensing 1 microliter of 5 mM ATP into all wells. Well fluorescence was read after 1 hour of incubation at 25 degrees Celsius on the Viewlux (Perkin-Elmer).

Prior to inhibition calculations the ratio between RFU values obtained at t0 (RFU_t0) and t60 (RFU_t60), named Ratio_RFU, was calculated as follows:

Ratio_RFU = RFU_t60 / RFU_t0

The percent inhibition for each well was then calculated as follows:

Percent inhibition = ( test_compound_Ratio_RFU - negative_control_Ratio_RFU ) / ( positive_control_Ratio_RFU - negative_control_Ratio_RFU ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Negative_Control is defined as wells containing DMSO.
Positive_Control is defined as wells containing Thioflavine S.

A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-16, for inactive 16-0.

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)
1536-well plates (Greiner, part 789173)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (10.9μM**)Normalized percent inhibition of the confirmation assay at a compound concentration of 10.9 micromolar.Float%
2Standard deviationStandard deviation of the confirmation assay derived from the normalized percent inhibition of the triplicate data for each compound.Float

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

Data Table (Concise)
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