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BioAssay: AID 489000

Late stage results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Fluorescence polarization-based biochemical dose response assay for EBNA-1 inhibitors

Name: Late stage results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Fluorescence polarization-based biochemical dose response assay for EBNA-1 inhibitors. ..more
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
Inactive(29)
 
 
 Tested Substances
 Tested Substances
All(30)
 
 
Active(1)
 
 
Inactive(29)
 
 
AID: 489000
Data Source: The Scripps Research Institute Molecular Screening Center (EBNA1_INH_FP_1536_3XIC50 MDRUN Round 0)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-11-08
Hold-until Date: 2011-11-04
Modify Date: 2011-11-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
1950Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1).Screeningdepositor-specified cross reference: Primary screen (EBNA-1 inhibitors in singlicate)
1955Summary of probe development efforts to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1).Summarydepositor-specified cross reference: Summary (EBNA-1 inhibitors)
2292Fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1).Screeningdepositor-specified cross reference: Confirmation (EBNA-1 inhibitors in triplicate)
2328Counterscreen for inhibitors of EBNA-1: fluorescence polarization-based biochemical high throughput assay for inhibitors of the Epstein-Barr virus-encoded protein, ZTA, in triplicate.Screeningdepositor-specified cross reference: Dose response counterscreen (ZTA inhibitors in triplicate)
2377Counterscreen for inhibitors of EBNA-1: fluorescence polarization-based biochemical high throughput dose response assay to identify inhibitors of the Epstein-Barr virus-encoded protein, ZTAConfirmatorydepositor-specified cross reference: Counterscreen (ZTA inhibitors in triplicate)
2381Fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1)Confirmatorydepositor-specified cross reference: Dose response (EBNA-1 inhibitors in triplicate)
2234Counterscreen for inhibitors of EBNA-1: fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of the Epstein-Barr virus-encoded protein, ZTA.Screeningsame project related to Summary assay
2361Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the Epstein-Barr virus-encoded protein, ZTAScreeningsame project related to Summary assay
2362Counterscreen for inhibitors of ZTA: fluorescence polarization-based biochemical high throughput assay to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1)Screeningsame project related to Summary assay
488998Late stage counterscreen results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): fluorescence polarization-based biochemical dose response assay for inhibitors of the Epstein-Barr virus-encoded protein, ZTAConfirmatorysame project related to Summary assay
493077Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Fluorescence polarization-based biochemical dose response assay for inhibitors of the Epstein-Barr virus-encoded protein, ZTAConfirmatorysame project related to Summary assay
493079Late stage assay provider results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Fluorescence polarization-based biochemical dose response assay for EBNA-1 inhibitorsConfirmatorysame project related to Summary assay
588498qHTS profiling assay for firefly luciferase inhibitor/activator using purifed enzyme and Km concentrations of substrates (counterscreen for the Campaign to Identify EBNA-1 Inhibitors project).Confirmatorysame project related to Summary assay
588762Late stage assay provider results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Cell-based luciferase-based dose response assay for EBNA-1 inhibitorsConfirmatorysame 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: Paul Lieberman, Wistar Institute
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: I R21 NS063906-01
Grant Proposal PI: Paul Lieberman, Wistar Institute
External Assay ID: EBNA1_INH_FP_1536_3XIC50 MDRUN Round 0

Name: Late stage results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Fluorescence polarization-based biochemical dose response assay for EBNA-1 inhibitors.

Description:

During each cell cycle in eukaryotes, the genome must be completely replicated and this replication must begin at the correct time and site (initiation site or origin) (1). Pathogenic viruses often take advantage of this cellular precision to maintain replication of their own genome. The Epstein-Barr virus (EBV) is an orally-transmitted herpesvirus associated with numerous human neoplasms (2) that infects over 90% of the world's population (3). Latent EBV infection stimulates B cell proliferation in vitro, and can lead to B cell transformation (4). Following infection, the EBV genome is maintained in the host cell as a plasmid that is replicated by machinery comprised of several host proteins and a sole EBV protein, the EBV nuclear antigen 1 (EBNA-1). This EBNA-1 protein is required for replication of the EBV DNA genome. EBNA-1 is a DNA-binding protein that binds to an EBV sequence called the viral origin of replication plasmid, OriP (5). Studies demonstrating a role for EBV in Burkitt's lymphoma and Hodgkin's disease (6), multiple sclerosis (7), lupus (8), infectious mononucleosis (9), and nasopharyngeal carcinoma (10), combined with the recent discovery that EBNA-1 induces DNA double strand breaks (11), suggest that inhibitors of EBNA-1 may serve useful for understanding virus-cell interactions, virus-mediated cellular transformation, and as therapies for EBV-associated pathologies.

References:

1. Norseen, J, Thomae, A, Sridharan, V, Aiyar, A, Schepers, A and Lieberman, PM, RNA-dependent recruitment of the origin recognition complex. EMBO J, 2008. 27(22): p. 3024-35.
2. Carbone, A, Gloghini, A and Dotti, G, EBV-associated lymphoproliferative disorders: classification and treatment. Oncologist, 2008. 13(5): p. 577-85.
3. Schulz, TF and Cordes, S, Is the Epstein-Barr virus EBNA-1 protein an oncogene? Proc Natl Acad Sci U S A, 2009. 106(7): p. 2091-2.
4. Thorley-Lawson, DA and Babcock, GJ, A model for persistent infection with Epstein-Barr virus: the stealth virus of human B cells. Life Sci, 1999. 65(14): p. 1433-53.
5. Kirchmaier, AL and Sugden, B, Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus. J Virol, 1997. 71(3): p. 1766-75.
6. Hammerschmidt, W and Sugden, B, Epstein-Barr virus sustains Burkitt's lymphomas and Hodgkin's disease. Trends Mol Med, 2004. 10(7): p. 331-6.
7. Lunemann, JD, Kamradt, T, Martin, R and Munz, C, Epstein-barr virus: environmental trigger of multiple sclerosis? J Virol, 2007. 81(13): p. 6777-84.
8. Harley, JB, Harley, IT, Guthridge, JM and James, JA, The curiously suspicious: a role for Epstein-Barr virus in lupus. Lupus, 2006. 15(11): p. 768-77.
9. Vetsika, EK and Callan, M, Infectious mononucleosis and Epstein-Barr virus. Expert Rev Mol Med, 2004. 6(23): p. 1-16.
10. Raab-Traub, N, Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol, 2002. 12(6): p. 431-41.
11. Gruhne, B, Sompallae, R, Marescotti, D, Kamranvar, SA, Gastaldello, S and Masucci, MG, The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species. Proc Natl Acad Sci U S A, 2009. 106(7): p. 2313-8.

Keywords:

late stage, powders, purchased, synthesized, EBNA-1, EBNA1, EBNA, Epstein-Barr virus, EBV, herpesvirus, DNA virus, human herpesvirus 4, HHV4, lymphoma, cancers, inhibitors, inhibition, antagonists, fluorescence polarization, FP, dose response, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN, Kansas, KU, Kansas University Specialized Chemistry Center, KUSCC.
Protocol
Assay Overview:
The purpose of this assay is to determine dose response curves for powder samples of compounds identified as possible EBNA-1 inhibitor probe candidates. This assay assesses the ability of compounds to inhibit the binding of EBNA-1 protein to DNA. In this biochemical assay, recombinant EBNA-1 DNA binding domain (DBD) (aa 448-610) purified from E. coli is incubated with a Cyanine (Cy5)-labeled 36-bp hairpin DNA oligonucleotide probe in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. As designed, compounds that act as EBNA-1 inhibitors will prevent EBNA-DNA binding, thereby increasing the proportion of free DNA in the well, resulting in low fluorescence polarization in the well. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 148 uM.
Protocol Summary:
Prior to the start of the assay, Assay Buffer (20 mM Tris HCl, pH 7.4; 200 mM NaCl; 1 mM DTT; 10 ug/ml BSA; 10 nM Cy5 DNA probe) containing 246 nM of EBNA-1 protein was prepared and incubated for 30 minutes at room temperature. Next, 4.0 uL of Assay Buffer were dispensed into 1536-well microtiter plates.
The assay started by dispensing 60 nL of test compound in DMSO or DMSO alone (1.5% final concentration) to the appropriate wells. Plates were then centrifuged and incubated for 1 hour at room temperature.
Fluorescence polarization was read on an Envision microplate reader (PerkinElmer, Turku, Finland) using a Cy5 FP filter set (Excitation = 620 nm, Emission = 688 nm) and a Cy5 dichroic mirror. Fluorescence polarization was read using 50 flashes per well.
The well Fluorescence Polarization (FP) value was calculated from the parallel (S) and perpendicular (P) polarization values using the following formula:
FP = (S - P) / (S + P).
The percent inhibition for each compound was calculated as follows:
% inhibition = ( Test_Compound - median_Low_Control ) / ( median_High_Control - median_Low_Control )* 100
Where:
Test_Compound is defined as wells containing EBNA-1 in the presence of test compound.
Low_Control is defined as wells containing EBNA-1.
High_Control is defined as wells containing no EBNA-1.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies 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. In cases where the highest concentration tested (i.e. 148 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 148 uM.
PubChem Activity Outcome and Score:
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, 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 89-0.
List of Reagents:
Recombinant EBNA-1 (supplied by Assay Provider)
Cy5 DNA probe (supplied by Assay Provider)
Tris HCl (Sigma, part T1503)
NaCl (Fisher, part S640-10)
DTT (Sigma, part D9779)
BSA (Calbiochem, part 126609)
1536-well plates (Corning, part 7261)
Comment
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, and 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 Kansas University Specialized Chemistry Center.
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
1QualifierActivity Qualifier identifies 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
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 0.008 uM [1] (0.008μM**)Value of % inhibition at 0.008 micromolar inhibitor concentration; replicate one.Float%
12Inhibition at 0.008 uM [2] (0.008μM**)Value of % inhibition at 0.008 micromolar inhibitor concentration; replicate two.Float%
13Inhibition at 0.008 uM [3] (0.008μM**)Value of % inhibition at 0.008 micromolar inhibitor concentration; replicate three.Float%
14Inhibition at 0.023 uM [1] (0.023μM**)Value of % inhibition at 0.023 micromolar inhibitor concentration; replicate one.Float%
15Inhibition at 0.023 uM [2] (0.023μM**)Value of % inhibition at 0.023 micromolar inhibitor concentration; replicate two.Float%
16Inhibition at 0.023 uM [3] (0.023μM**)Value of % inhibition at 0.023 micromolar inhibitor concentration; replicate three.Float%
17Inhibition at 0.068 uM [1] (0.068μM**)Value of % inhibition at 0.068 micromolar inhibitor concentration; replicate one.Float%
18Inhibition at 0.068 uM [2] (0.068μM**)Value of % inhibition at 0.068 micromolar inhibitor concentration; replicate two.Float%
19Inhibition at 0.068 uM [3] (0.068μM**)Value of % inhibition at 0.068 micromolar inhibitor concentration; replicate three.Float%
20Inhibition at 0.203 uM [1] (0.203μM**)Value of % inhibition at 0.203 micromolar inhibitor concentration; replicate one.Float%
21Inhibition at 0.203 uM [2] (0.203μM**)Value of % inhibition at 0.203 micromolar inhibitor concentration; replicate two.Float%
22Inhibition at 0.203 uM [3] (0.203μM**)Value of % inhibition at 0.203 micromolar inhibitor concentration; replicate three.Float%
23Inhibition at 0.608 uM [1] (0.608μM**)Value of % inhibition at 0.608 micromolar inhibitor concentration; replicate one.Float%
24Inhibition at 0.608 uM [2] (0.608μM**)Value of % inhibition at 0.608 micromolar inhibitor concentration; replicate two.Float%
25Inhibition at 0.608 uM [3] (0.608μM**)Value of % inhibition at 0.608 micromolar inhibitor concentration; replicate three.Float%
26Inhibition at 1.8 uM [1] (1.8μM**)Value of % inhibition at 1.8 micromolar inhibitor concentration; replicate one.Float%
27Inhibition at 1.8 uM [2] (1.8μM**)Value of % inhibition at 1.8 micromolar inhibitor concentration; replicate two.Float%
28Inhibition at 1.8 uM [3] (1.8μM**)Value of % inhibition at 1.8 micromolar inhibitor concentration; replicate three.Float%
29Inhibition at 5.5 uM [1] (5.5μM**)Value of % inhibition at 5.5 micromolar inhibitor concentration; replicate one.Float%
30Inhibition at 5.5 uM [2] (5.5μM**)Value of % inhibition at 5.5 micromolar inhibitor concentration; replicate two.Float%
31Inhibition at 5.5 uM [3] (5.5μM**)Value of % inhibition at 5.5 micromolar inhibitor concentration; replicate three.Float%
32Inhibition at 16.4 uM [1] (16.4μM**)Value of % inhibition at 16.4 micromolar inhibitor concentration; replicate one.Float%
33Inhibition at 16.4 uM [2] (16.4μM**)Value of % inhibition at 16.4 micromolar inhibitor concentration; replicate two.Float%
34Inhibition at 16.4 uM [3] (16.4μM**)Value of % inhibition at 16.4 micromolar inhibitor concentration; replicate three.Float%
35Inhibition at 49.3 uM [1] (49.3μM**)Value of % inhibition at 49.3 micromolar inhibitor concentration; replicate one.Float%
36Inhibition at 49.3 uM [2] (49.3μM**)Value of % inhibition at 49.3 micromolar inhibitor concentration; replicate two.Float%
37Inhibition at 49.3 uM [3] (49.3μM**)Value of % inhibition at 49.3 micromolar inhibitor concentration; replicate three.Float%
38Inhibition at 147.8 uM [1] (147.8μM**)Value of % inhibition at 147.8 micromolar inhibitor concentration; replicate one.Float%
39Inhibition at 147.8 uM [2] (147.8μM**)Value of % inhibition at 147.8 micromolar inhibitor concentration; replicate two.Float%
40Inhibition at 147.8 uM [3] (147.8μM**)Value of % inhibition at 147.8 micromolar inhibitor concentration; replicate three.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: I R21 NS063906-01

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