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BioAssay: AID 588398

TES1 - eGFP vs TES2 -dsRED Pathway Differentiation Measured in Cell-Based System Using Imaging - 2122-04_Inhibitor_Dose_CherryPick_Activity

NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, cytotoxicity, EBV-transformed TES 1 - eGFP and TES2 - dsRED ..more
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 Tested Compounds
 Tested Compounds
All(1240)
 
 
Active(113)
 
 
Inactive(1030)
 
 
Inconclusive(97)
 
 
 Tested Substances
 Tested Substances
All(1241)
 
 
Active(113)
 
 
Inactive(1031)
 
 
Inconclusive(97)
 
 
AID: 588398
Data Source: Broad Institute (2122-04_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-09-22

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 113
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
504861Viability counterscreen of potential LMP-1 inhibitors in HEK293 cell background Measured in Cell-Based System Using Plate Reader - 2122-02_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame 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
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, cytotoxicity, EBV-transformed TES 1 - eGFP and TES2 - dsRED

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. This assay uses a dual color reporter system in HEK293T cells stably transfected with a contruct containing EGFP driven by a TES-1 responsive promoter and a dsRED reporter driven by a Tet-On tetracycline responsive element.

Expected Outcome:
Compounds that are inhibiting the eGFP are inhibiting the TES pathway, while compounds inhibiting the TET-ON dsRed signal are not specific for NF-kB. Compounds that are of interest will selectively inhibit the eGFP signal and not the dsRED production and will be carried forward. A window of at least 2-fold is desired. Partial inhibition of eGFP in this assay are of interest, as they could be acting through just one of the canonical/non-canonical pathways.
Protocol
LMP1 - Secondary Screen 2 Secondary Screen Protocol
(eGFP/dsRED Assay )

Day 0, cell grown in t175 TC flask to 95% confluence to yield 30 Million (TrypLE phenol free) and resuspended to dispensing at 125,000 cells / mL of phenol free DMEM

Day 1, plate cells 5000 per well in 40 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 24 hours.

Day 2, add 10 uL per well of stimulant (3ug/mL doxycycline and 3uM 4-hydroxytamxoifen in phenol free DMEM medium) with a Combi multidrop (Thermo);
add 100 nL 3.75 mM compound library into 50 uL assay volume in Black Clear bottom Corning 8816BC barcoded 384 well plates using a pin tool (CyBio). Final compound library concentration was 9.4 uM. MG132 (Calbiochem 474790), a proteasome inhibitor that blocks degradation of NF-kappaB inhibitor Ikappa-Balpha, was added to positive control wells to a final concentration of 12.5 uM.
Incubate 48 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.

Day 4, remove plate from incubator ; Read on Acumen eX3 with 488 excitation laser and detect with green and red filters, gated at 2SD and 562 voltage. <>
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 in the eGFP channel only, with a 2-fold less potent response in the dsRED channel
The compounds were measured in multiple channels; each instance is called a 'TEST'. TEST_1 corresponds to the dsRED channel, and TEST_2 corresponds to the eGFP channel. For each channel, the following analysis was applied:

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 (2)(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.

DATA AGGREGATION:
Once the data for each channel was processed, the channel name was appended to all column headers in that channel's data set. The individual channel results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when AC50 of the dsRED channel (TEST1) is greater than 2 * AC50 of the eGFP channel (TEST2), or dsRED (TEST1) was inactive and eGFP (TEST2) < AC_limit;
b. '1' (inactive) when all channel outcomes were '1' (inactive), or when AC50 of the dsRED channel (TEST1) is less than 2 * AC50 of the eGFP channel (TEST2).
c. '3' (inconclusive) when one or more channel outcomes were inconclusive.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the value of the eGFP channel (TEST2);
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE for active compounds is equal to the activity score of the eGFP channel (TEST2).

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.
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
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.009uM_(%)_TEST1 (0.009μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.0285uM_(%)_TEST1 (0.0285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.08uM_(%)_TEST1 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.235uM_(%)_TEST1 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.75uM_(%)_TEST1 (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_6.8uM_(%)_TEST1 (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_19.5uM_(%)_TEST1 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
26ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
27ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
28AC50_Qualifier_TEST2'>','=', or '<'String
29AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
30pAC50_M_TEST2Equal to -1*log10(AC50)String
31Hill_Slope_TEST2The slope at AC50Float
32S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
33Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
34Num_Points_TEST2The number of data points used to generate the plotInteger
35Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
36Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
37Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
38Activity_at_0.009uM_(%)_TEST2 (0.009μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_0.0285uM_(%)_TEST2 (0.0285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.08uM_(%)_TEST2 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.235uM_(%)_TEST2 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.75uM_(%)_TEST2 (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_2.1uM_(%)_TEST2 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_6.8uM_(%)_TEST2 (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_19.5uM_(%)_TEST2 (19.5μM**)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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