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BioAssay: AID 463085

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 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 AlphaScreen assay to identify inhibitors of HCV core dimerization. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(1)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(1)
 
 
Inactive(2)
 
 
AID: 463085
Data Source: The Scripps Research Institute Molecular Screening Center (HCV-ALPHASCREEN_INH_LUMI_3XIC50 MDCSRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-08-06
Hold-until Date: 2011-08-04
Modify Date: 2012-09-17

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
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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 Primary screen (HCV dimerization inhibitors in singlicate)
1911Summary of probe development efforts to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerizationsummary Summary (HCV dimerization inhibitors)
2152TR-FRET-based biochemical high-throughput confirmation assay for inhibitors of Hepatitis C Virus (HCV) core protein dimerization.screening 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 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 Late stage dose response (HCV core 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
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
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
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
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
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
651577Late 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 dimerizationconfirmatory1
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
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

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 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 assay is to determine whether powder samples of compounds identified as possible HCV core probe candidates can 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 pH7.5, 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 100-100, and for inactive compounds 17-1.

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 fluorescence. All test compound concentrations reported above and below are nominal.
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
2Inhibition at 0.05 uM [1] (0.05μM**)Value of %inhibition at 0.05 uM inhibitor concentration; replicate one.Float%
3Inhibition at 0.05 uM [2] (0.05μM**)Value of %inhibition at 0.05 uM inhibitor concentration; replicate two.Float%
4Inhibition at 0.05 uM [3] (0.05μM**)Value of %inhibition at 0.05 uM inhibitor concentration; replicate three.Float%
5Inhibition at 0.1 uM [1] (0.1μM**)Value of %inhibition at 0.1 uM inhibitor concentration; replicate one.Float%
6Inhibition at 0.1 uM [2] (0.1μM**)Value of %inhibition at 0.1 uM inhibitor concentration; replicate two.Float%
7Inhibition at 0.1 uM [3] (0.1μM**)Value of %inhibition at 0.1 uM inhibitor concentration; replicate three.Float%
8Inhibition at 0.5 uM [1] (0.5μM**)Value of %inhibition at 0.5 uM inhibitor concentration; replicate one.Float%
9Inhibition at 0.5 uM [2] (0.5μM**)Value of %inhibition at 0.5 uM inhibitor concentration; replicate two.Float%
10Inhibition at 0.5 uM [3] (0.5μM**)Value of %inhibition at 0.5 uM inhibitor concentration; replicate three.Float%
11Inhibition at 1 uM [1] (1μM**)Value of %inhibition at 1 uM inhibittor concentration; replicate one.Float%
12Inhibition at 1 uM [2] (1μM**)Value of %inhibition at 1 uM inhibittor concentration; replicate two.Float%
13Inhibition at 1 uM [3] (1μM**)Value of %inhibition at 1 uM inhibittor concentration; replicate three.Float%
14Inhibition at 10 uM [1] (10μM**)Value of %inhibition at 10 uM inhibitor concentration; replicate one.Float%
15Inhibition at 10 uM [2] (10μM**)Value of %inhibition at 10 uM inhibitor concentration; replicate two.Float%
16Inhibition at 10 uM [3] (10μM**)Value of %inhibition at 10 uM inhibitor concentration; replicate three.Float%
17Inhibition at 15 uM [1] (15μM**)Value of %inhibition at 15 uM inhibitor concentration; replicate one.Float%
18Inhibition at 15 uM [2] (15μM**)Value of %inhibition at 15 uM inhibitor concentration; replicate two.Float%
19Inhibition at 15 uM [3] (15μM**)Value of %inhibition at 15 uM inhibitor concentration; replicate three.Float%
20Inhibition at 20 uM [1] (20μM**)Value of %inhibition at 20 uM inhibitor concentration; replicate one.Float%
21Inhibition at 20 uM [2] (20μM**)Value of %inhibition at 20 uM inhibitor concentration; replicate two.Float%
22Inhibition at 20 uM [3] (20μM**)Value of %inhibition at 20 uM inhibitor concentration; replicate three.Float%
23Inhibition at 50 uM [1] (50μM**)Value of %inhibition at 50 uM inhibitor concentration; replicate one.Float%
24Inhibition at 50 uM [2] (50μM**)Value of %inhibition at 50 uM inhibitor concentration; replicate two.Float%
25Inhibition at 50 uM [3] (50μM**)Value of %inhibition at 50 uM inhibitor concentration; replicate three.Float%
26Inhibition at 75 uM [1] (75μM**)Value of %inhibition at 75 uM inhibitor concentration; replicate one.Float%
27Inhibition at 75 uM [2] (75μM**)Value of %inhibition at 75 uM inhibitor concentration; replicate two.Float%
28Inhibition at 75 uM [3] (75μM**)Value of %inhibition at 75 uM inhibitor concentration; replicate three.Float%
29Inhibition at 100 uM [1] (100μM**)Value of %inhibition at 100 uM inhibitor concentration; replicate one.Float%
30Inhibition at 100 uM [2] (100μM**)Value of %inhibition at 100 uM inhibitor concentration; replicate two.Float%
31Inhibition at 100 uM [3] (100μM**)Value of %inhibition at 100 uM inhibitor concentration; replicate three.Float%

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

Data Table (Concise)
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