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BioAssay: AID 651577

Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Luminescence-based biochemical dose response AlphaScreen assay to identify inhibitors of HCV core dimerization

Name: Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Luminescence-based biochemical dose response AlphaScreen assay to identify inhibitors of HCV core dimerization. ..more
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 Tested Compounds
 Tested Compounds
All(14)
 
 
Probe(1)
 
 
Active(8)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(14)
 
 
Probe(1)
 
 
Active(8)
 
 
Inactive(6)
 
 
AID: 651577
Data Source: The Scripps Research Institute Molecular Screening Center (HCV-ALPHASCREEN_INH_LUMI_3XIC50 MDCSRUN Round 1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-09-11
Hold-until Date: 2013-06-25
Modify Date: 2013-06-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 1    Active: 8
Related Experiments
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AIDNameTypeProbeComment
1899TR-FRET-based primary biochemical high-throughput screening assay to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerizationScreening depositor-specified cross reference: Primary screen (HCV dimerization inhibitors in singlicate)
1911Summary of probe development efforts to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerizationSummary depositor-specified cross reference: Summary (HCV dimerization inhibitors)
2152TR-FRET-based biochemical high-throughput confirmation assay for inhibitors of Hepatitis C Virus (HCV) core protein dimerization.Screening depositor-specified cross reference: Confirmation screen (HCV dimerization inhibitors in triplicate)
2159TR-FRET-based biochemical high-throughput dose response assay to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization.Confirmatory depositor-specified cross reference: Dose response (HCV dimerization inhibitors in triplicate)
2488Late stage results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: TR-FRET-based biochemical dose response assay for HCV core inhibitorsConfirmatory depositor-specified cross reference: Late stage dose response (HCV core inhibitors in triplicate)
463085Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Luminescence-based biochemical AlphaScreen assay to identify inhibitors of HCV core dimerizationConfirmatory depositor-specified cross reference: Late stage Alpha Screen dose response (HCV core dimerization inhibitors in triplicate)
485271Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: fluorescence-based cell-based quantitative PCR assay to identify inhibitors of HCV infectivityConfirmatory depositor-specified cross reference: Late stage counterscreen (HCV infectivity inhibitors in triplicate)
485280Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Absorbance-based cell-based assay to identify compounds that are cytotoxic to Huh-7.5 cellsConfirmatory depositor-specified cross reference: Late stage counterscreen (Cytotoxicity in triplicate)
624406Late stage results from the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Cell-based radioligand binding assay to determine the binding affinities for selected transporters, receptors, and GPCRsOther depositor-specified cross reference: Late stage (selected transporters, receptors, and GPCRs binding affinities)
651574Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Fluorescence-based cell-based Quantitative Polymerase Chain Reaction (QPCR) assay to identify inhibitors of HCV infectivity (2 timepoints)Other1 same project related to Summary assay
651575Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Fluorescence-based cell-based Quantitative Polymerase Chain Reaction (QPCR) assay to identify inhibitors of HCV infectivityConfirmatory1 same project related to Summary assay
651576Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Luminescence-based biochemical AlphaScreen assay to identify inhibitors of HCV core dimerization (%INH 15uM)Other1 same project related to Summary assay
651583Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Absorbance-based cell-based assay to identify compounds that are cytotoxic to Huh-7.5 cellsConfirmatory same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: A.D. Strosberg, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-X01-MH085709-01
Grant Proposal PI: A.D. Strosberg, TSRI
External Assay ID: HCV-ALPHASCREEN_INH_LUMI_3XIC50 MDCSRUN Round 1

Name: Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Luminescence-based biochemical dose response AlphaScreen assay to identify inhibitors of HCV core dimerization.

Description:

The 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 most N-terminal 21kDa protein of this HCV polyprotein is the HCV core (C) protein, which is a highly basic, RNA-binding structural protein essential for assembly and packaging of the viral genome (2). Core protein is cleaved by a host peptidase and anchored to the host cell endoplasmic reticulum, where it undergoes further processing into its mature form (3). The N terminal portion of this mature C protein mediates viral assembly through homodimerization and formation of higher order complexes with viral RNA to form the nucleocapsid, while the hydrophobic C terminal interacts with envelope glycoproteins to form the infectious particle (4). The conserved nature of the HCV protein and absence of a vaccine to prevent HCV infection (5), along with studies demonstrating that C protein contributes to host cell oncogenesis (6), apoptosis inhibition (7), and suppression of host T cell responses (8), support a role for core protein as a major pathogenic component of HCV. The identification of specific inhibitors of HCV core dimerization will provide valuable tools for inhibiting HCV assembly without host cell effects.

References:

1. Hoofnagle, J.H., Course and outcome of hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. s21-s29.
2. Lin, C., Lindenbach, B.D., Pragai, B.M., McCourt, D.W., and Rice, C.M., Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol, 1994. 68(8): p. 5063-73.
3. Moradpour, D. and Blum, H.E., A primer on the molecular virology of hepatitis C. Liver Int, 2004. 24(6): p. 519-25.
4. Majeau, N., Gagne, V., Boivin, A., Bolduc, M., Majeau, J.A., Ouellet, D., and Leclerc, D., The Nterminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation. J Gen Virol, 2004. 85(Pt 4): p. 971-81.
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. Ray, R.B., Lagging, L.M., Meyer, K., and Ray, R., Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J Virol, 1996. 70(7): p. 4438-43.
7. Marusawa, H., Hijikata, M., Chiba, T., and Shimotohno, K., Hepatitis C virus core protein inhibits Fasand tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation. J Virol, 1999. 73(6): p. 4713-20.
8. Large, M.K., Kittlesen, D.J., and Hahn, Y.S., Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. J Immunol, 1999. 162(2): p. 931- 8.
9. Kota S, Coito C, Mousseau G, Lavergne JP, Strosberg AD. Peptide inhibitors of hepatitis C virus core oligomerization and virus production. J Gen Virol. 2009 Jun;90(Pt 6):1319-28.
10. Peppard J, Glickman F, He Y, Hu SI, Doughty J, Goldberg R. Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen ). J Biomol Screen. 2003 Apr;8(2):149-56.

Keywords:

late stage, late stage AID, powders, purchased, HCV, core protein, core 106, core, hepatitis, hepatitis C, RNA virus, protein-protein interaction, dimerization, dose response, biochemical, AlphaScreen, alpha, liver, hepatocytes, assay provider, inhibitor, inhibition, inhibit, luminescence, viability, 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 dose response assay is to determine IC50 values for powder samples of compounds identified as possible HCV core probe candidates. This assay determines the ability of compounds to disrupt the dimerization of HCV Core molecules (9). This assay employs the AlphaScreen technology, a secondary Amplified Luminescent Proximity Homogeneous Assay. This assay is run by the assay provider. Compounds were tested in triplicate in a 10-point series starting at a nominal test concentration of 50 uM.
Protocol Summary:
AlphaScreen is based on the use of photoactive donor and acceptor beads that recognize specific tags on interacting proteins (10). Core106 dimerization was confirmed using AlphaScreen technology in which a core106 protein domain was tagged with either Glutathione-S-transferase (GST) tag or a Flag peptide tag. The untagged core106 protein domain was used as a model competitor in the assay. The proteins were diluted to working concentrations in 'protein buffer' (100 mM HEPES pH 7.5, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS, 10% glycerol). The donor and acceptor beads were diluted to working concentrations in 'bead buffer' (20 mM HEPES pH 7.5, 125 mM NaCl, 0.1% BSA, 0.1% CHAPS). GST-tagged core106 (150 nM) was incubated with 150 nM of Flag-tagged core106 for 1 hour at room temperature. Anti-Flag acceptor beads were added to the proteins at a final concentration of 20 ug/ml and incubated for 1 hour at room temperature. Then Glutathione donor beads were added to the proteins at a final concentration of 20 ug/ml and incubated for 1 hour. The assays were executed in a white 384 well Packard opti plate and were read on Perkin Elmer Envision.
The percent inhibition for each compound is reported as the average and the standard deviation of three replicate wells, calculated as follows:
%_Inhibition = ( ( Uninhibited - Test_Compound ) / Uninhibited ) * 100
Where:
Test_Compound is defined as wells containing test compound.
Uninhibited or 0% inhibition control is defined as wells containing only GST-core106 and Flag-core106.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was plotted using GraphPad Prism (GraphPad Software 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.
PubChem Activity Score and Outcome:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value > 50% at any test concentration was assigned an activity score greater than zero. 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 100-83, and for inactive compounds 77-1.
List of Reagents:
GST-core106 (supplied by Assay Provider)
Flag-core106 (supplied by Assay Provider)
Unlabelled core106 peptide (supplied by Assay Provider)
Glutathione coated donor beads (Perkin Elmer Lifesciences, part 6765300)
Anti-Flag antibody coated acceptor beads (Perkin Elmer Lifesciences, part AL112M)
384-well Opti plates (Perkin Elmer Lifesciences, part 6007299)
HEPES (Sigma part H3537)
EDTA (Invitrogen part 15575-020)
Glycerol (Fisher Scientific part BP-224-4)
Bovine Serum Albumin (Sigma part A7030)
CHAPS (Anatrace part C316)
DTT (Sigma part 43817)
Sodium Chloride (Fisher Scientific part BP-358-212)
Comment
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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 luminescence. All test compound concentrations reported are nominal.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
2Average Inhibition at 160 uM (160μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
3Standard Deviation [Average Inhibition at 160 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
4Inhibition at 160 uM [1] (160μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
5Inhibition at 160 uM [2] (160μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
6Inhibition at 160 uM [3] (160μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
7Average Inhibition at 40 uM (40μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
8Standard Deviation [Average Inhibition at 40 uM] The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
9Inhibition at 40 uM [1] (40μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
10Inhibition at 40 uM [2] (40μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
11Inhibition at 40 uM [3] (40μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
12Average Inhibition at 10 uM (40μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
13Standard Deviation [Average Inhibition at 10 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
14Inhibition at 10 uM [1] (10μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
15Inhibition at 10 uM [2] (10μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
16Inhibition at 10 uM [3] (10μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
17Average Inhibition at 2.5 uM (2.5μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
18Standard Deviation [Average Inhibition at 2.5 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
19Inhibition at 2.5 uM [1] (2.5μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
20Inhibition at 2.5 uM [2] (2.5μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
21Inhibition at 2.5 uM [3] (2.5μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
22Average Inhibition at 0.625 uM (0.625μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
23Standard Deviation [Average Inhibition at 0.625 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
24Inhibition at 0.625 uM [1] (0.625μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
25Inhibition at 0.625 uM [2] (0.625μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
26Inhibition at 0.625 uM [3] (0.625μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
27Average Inhibition at 0.156 uM (0.156μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
28Standard Deviation [Average Inhibition at 0.156 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
29Inhibition at 0.156 uM [1] (0.156μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
30Inhibition at 0.156 uM [2] (0.156μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
31Inhibition at 0.156 uM [3] (0.156μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
32Average Inhibition at 0.039 uM (0.039μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
33Standard Deviation [Average Inhibition at 0.039 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
34Inhibition at 0.039 uM [1] (0.039μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
35Inhibition at 0.039 uM [2] (0.039μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
36Inhibition at 0.039 uM [3] (0.039μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
37Average Inhibition at 0.0097 uM (0.0097μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
38Standard Deviation [Average Inhibition at 0.0097 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
39Inhibition at 0.0097 uM [1] (0.0097μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
40Inhibition at 0.0097 uM [2] (0.0097μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
41Inhibition at 0.0097 uM [3] (0.0097μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
42Average Inhibition at 0.0024 uM (0.0024μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
43Standard Deviation [Average Inhibition at 0.0024 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
44Inhibition at 0.0024 uM [1] (0.0024μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
45Inhibition at 0.0024 uM [2] (0.0024μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
46Inhibition at 0.0024 uM [3] (0.0024μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
47Average Inhibition at 0.0006 uM (0.0006μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
48Standard Deviation [Average Inhibition at 0.0006 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float
49Inhibition at 0.0006 uM [1] (0.0006μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [1].Float%
50Inhibition at 0.0006 uM [2] (0.0006μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [2].Float%
51Inhibition at 0.0006 uM [3] (0.0006μM**)The percentage of HCV Core dimerization inhibition at the indicated dose of test compound; replicate [3].Float%
52Average Inhibition at 0.00015 uM (0.00015μM**)The average percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float%
53Standard Deviation [Average Inhibition at 0.00015 uM]The standard deviation of the percentage of HCV Core dimerization inhibition at the indicated dose of test compound.Float

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1-X01-MH085709-01

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