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BioAssay: AID 504861

Viability counterscreen of potential LMP-1 inhibitors in HEK293 cell background Measured in Cell-Based System Using Plate Reader - 2122-02_Inhibitor_Dose_CherryPick_Activity

NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, HEK 293T cytotoxicity ..more
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 Tested Compounds
 Tested Compounds
All(1240)
 
 
Active(287)
 
 
Inactive(922)
 
 
Inconclusive(32)
 
 
 Tested Substances
 Tested Substances
All(1241)
 
 
Active(287)
 
 
Inactive(922)
 
 
Inconclusive(32)
 
 
AID: 504861
Data Source: Broad Institute (2122-02_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-06-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 287
Related Experiments
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AIDNameTypeComment
504586Broad Institute Epstein-Barr LMP-1 Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
504558Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
504882Lymphoblastoid Cells (LCL) Cytotoxicity Secondary Assay Measured in Cell-Based System Using Plate Reader - 2122-03_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatorysame project related to Summary assay
588343Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
588398TES1 - eGFP vs TES2 -dsRED Pathway Differentiation Measured in Cell-Based System Using Imaging - 2122-04_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
624359Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624360Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624361Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624365Viability counterscreen of potential LMP-1 inhibitors in HepG2 cell line Measured in Cell-Based System Using Plate Reader - 2122-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624367Lymphoblastoid Cells (LCL) Cytotoxicity Secondary Assay Measured in Cell-Based System Using Plate Reader - 2122-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624369Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624372Viability counterscreen of potential LMP-1 inhibitors in HEK293 cell background Measured in Cell-Based System Using Plate Reader - 2122-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624373Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624374Viability counterscreen of potential LMP-1 inhibitors in HEK293 cell background Measured in Cell-Based System Using Plate Reader - 2122-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624375Lymphoblastoid Cells (LCL) Cytotoxicity Secondary Assay Measured in Cell-Based System Using Plate Reader - 2122-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624376Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
652043TNFalpha- eGFP -dsRED Canonical Pathway Differentiation Measured in Cell-Based System Using Flow Cytometry - 2122-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
652046IL-1beta eGFP -dsRED non-canonical Pathway Differentiation Measured in Cell-Based System Using Flow Cytometry - 2122-09_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords:
NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, HEK 293T cytotoxicity

Assay Overview:
Epstein-Barr Virus is a ubiquitous Herpesvirus that is an important cause of Hodgkin's Disease, other Lymphoproliferative Diseases, and Nasopharyngeal Carcinoma. EBV infection mimics NF-kB hyperactivation states present in many malignancies. The EBV oncoprotein LMP1 constitutively activates both canonical and noncanonical NF-kB pathways in a ligand-independent fashion. LMP1 is expressed in most EBV-associated lymphoproliferative and epithelial malignancies. LMP1 activates NF-kB via two cytoplasmic signaling domains. The membrane proximal "TES1" domain activates a non-canonical NF-kappaB pathway, while the membrane distal "TES2" domain activates canonical NF-kappaB.

The Counter Screen use un-transfected HEK293T cells from ATCC to counter screen for the cytoxicity of the at dose retested compounds from the primary screen.

Expected Outcome:
Compounds that are cytoxic will inhibit the signal. The compounds of interest will maintain at the signal of the neutral control (DMSO)
Protocol
LMP1 - Secondary Screen 1 counter Screen Protocol
(HEK 293T Cytotoxicity Assay )
Day 0, cell grown in Triple Flask (NUNC) to 95% confluence to yield 273 Million (TrypLE phenol free) and resuspended to dispensing at 150,000 cells / mL of phenol free DMEM
Day 1, plate cells 7,500 per well in 50 uL media (phenol red free DMEM/10% Tet Free FBS/Pen/Strep/L-Glutamine); incubate in standard TC conditions (5% CO2; 95% humidity, 37C) for 48 hours.
Day 2, add 100 nL 3.75 mM compound library into 50 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (HiRes Biosolutions). Final compound library concentration was BAY11-7082 (Calbiochem 196870), inhibits transcription factor NF-kappaB where 200 nL of 80 mM stock concentraiton at final concentration 16 uM.
Incubate 48 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 4, remove plate from incubator to cool for 15 minutes to room temperature; add 20 uL 50% Promega CellTiterGlo (diluted 1:1 with PBS pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.1 sec per well.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Assay Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.011uM_(%) (0.011μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.035uM_(%) (0.035μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.3uM_(%) (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.9uM_(%) (0.9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_2.85uM_(%) (2.85μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_8uM_(%) (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 2 R01 CA085180-06A1

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