Bookmark and Share
BioAssay: AID 623973

Late 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 activity

Name: Late 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 activity. ..more
_
   
 Tested Compounds
 Tested Compounds
All(31)
 
 
Active(13)
 
 
Inactive(18)
 
 
 Tested Substances
 Tested Substances
All(33)
 
 
Active(13)
 
 
Inactive(20)
 
 
AID: 623973
Data Source: The Scripps Research Institute Molecular Screening Center (HCV NS3_INH_ABS_96_IC50)
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
Target
BioActive Compounds: 13
Related Experiments
Show more
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
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 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
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_ABS_96_IC50

Name: Late 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 activity.

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.
8. Lanzetta,P.A., Alvarez,L.J., Reinach,P.S. and Candia,O.A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95-97.

Keywords:

late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, HCV, NS3, NS3 helicase, hepatitis, NS3h_1b(Con1), 1b(Con1), RNA virus, ATPase, malachite green, malachite green dye, molydbate, dose response, counterscreen, 96, assay provider, inhibitor, inhibition, inhibit, 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 verify enzyme activity 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 genotype 1b(Con1). The assay monitors the amount of ATP hydrolysis by NS3h_1b(Con1). A modified colorimetric "malachite green" assay was used (8). The hydrolysis of ATP releases an inorganic phosphate molecule that forms a complex with molybdate. This complex then forms a subsequent colored complex with the malachite green dye under acidic conditions and absorbance of the colored complex is read in a plate reader.

Protocol Summary:

Reactions contained 25 mM MOPS pH 6.5, 1.25 mM MgCl2 and 1 mM ATP in 30 uL total volume. The colorimetric reagent was prepared fresh by mixing 3 volumes 0.045% (w/v) malachite green, 1 volume 4.2% ammonium molybdate in 4N HCl, and 0.05 volume of 20% Tween 20. For SAR profiling, reactions were performed in 10% DMSO with 8 concentrations of each compound ranging from 200 uM to 1.6 uM in the absence of RNA with 50 uM of NS3h_1b(Con1). Reactions were initiated by adding ATP, incubated for 30 minutes at 23 C, and terminated by adding 200 ul of the malachite green reagent, followed by 30 uL of 35% sodium citrate. The color was allowed to develop for 30 minutes and absorbance at 630 nm was read. Percent inhibition was calculated by normalizing the data to reactions without enzyme (100% inhibition) and reactions with DMSO only (0% inhibition). Average IC50 values were then calculated from a normalized dose response curve of replicate assays using GraphPad Prism (v. 5).

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 100 uM were considered inactive. Compounds with an IC50 equal to or less than 100 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.

The PubChem Activity Score range for active compounds is 100-64, and for inactive compounds 32-0.

List of Reagents:

NS3 helicase fragment (supplied by Assay Provider)
MOPS (Fisher Scientific, part BP308-100)
ATP (Fisher Scientific, part BP413-25)
Magnesium Chloride (Fisher Scientific, part BP214-500)
Malachite Green (Acros, 41349-0250)
Ammonium Molybdate (Fisher Scientific, A674-500)
HCl (Fisher Scientific, A142-212)
Tween 20 (Fisher Scientific, BP337-500)
Assay Buffer (supplied by Assay Provider)
96-well plates (Corning Costar, clear, part 9017)
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 - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
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: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: spectrophotometry: absorbance
BAO: meta target: biological process target: viral genome replication
BAO: meta target: molecular target: protein target: enzyme regulator
BAO: version: 1.4b1090
From PubChem:
Assay Format: Biochemical
Result Definitions
Show more
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 average of the concentration at which 50 percent of the activity in the antagonist assay is observed (IC50); shown in micromolarFloatμM
3Standard ErrorStandard Error of the Average IC50. ND if not determined.Float
4Inhibition at 200 uM [1] (200μM**)Value of percent inhibition at 200 uM compound concentrationFloat%
5Inhibition at 100 uM [1] (100μM**)Value of percent inhibition at 100 uM compound concentrationFloat%
6Inhibition at 50 uM [1] (50μM**)Value of percent inhibition at 50 uM compound concentrationFloat%
7Inhibition at 25 uM [1] (25μM**)Value of percent inhibition at 25 uM compound concentrationFloat%
8Inhibition at 12.5 uM [1] (12.5μM**)Value of percent inhibition at 12.5 uM compound concentrationFloat%
9Inhibition at 6.25 uM [1] (6.25μM**)Value of percent inhibition at 6.25 uM compound concentrationFloat%
10Inhibition at 3.13 uM [1] (3.13μM**)Value of percent inhibition at 3.13 uM compound concentrationFloat%
11Inhibition at 1.56 uM [1] (1.56μM**)Value of percent inhibition at 1.56 uM compound concentrationFloat%
12Inhibition at 200 uM [2] (200μM**)Value of percent inhibition at 200 uM compound concentrationFloat%
13Inhibition at 100 uM [2] (100μM**)Value of percent inhibition at 100 uM compound concentrationFloat%
14Inhibition at 50 uM [2] (50μM**)Value of percent inhibition at 50 uM compound concentrationFloat%
15Inhibition at 25 uM [2] (25μM**)Value of percent inhibition at 25 uM compound concentrationFloat%
16Inhibition at 12.5 uM [2] (12.5μM**)Value of percent inhibition at 12.5 uM compound concentrationFloat%
17Inhibition at 6.25 uM [2] (6.25μM**)Value of percent inhibition at 6.25 uM compound concentrationFloat%
18Inhibition at 3.13 uM [2] (3.13μM**)Value of percent inhibition at 3.13 uM compound concentrationFloat%
19Inhibition at 1.56 uM [2] (1.56μM**)Value of percent inhibition at 1.56 uM compound concentrationFloat%
20Inhibition at 200 uM [3] (200μM**)Value of percent inhibition at 200 uM compound concentrationFloat%
21Inhibition at 100 uM [3] (100μM**)Value of percent inhibition at 100 uM compound concentrationFloat%
22Inhibition at 50 uM [3] (50μM**)Value of percent inhibition at 50 uM compound concentrationFloat%
23Inhibition at 25 uM [3] (25μM**)Value of percent inhibition at 25 uM compound concentrationFloat%
24Inhibition at 12.5 uM [3] (12.5μM**)Value of percent inhibition at 12.5 uM compound concentrationFloat%
25Inhibition at 6.25 uM [3] (6.25μM**)Value of percent inhibition at 6.25 uM compound concentrationFloat%
26Inhibition at 3.13 uM [3] (3.13μM**)Value of percent inhibition at 3.13 uM compound concentrationFloat%
27Inhibition at 1.56 uM [3] (1.56μM**)Value of percent inhibition at 1.56 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
PageFrom: