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BioAssay: AID 504558

Inhibitors of Epstein-Barr LMP1 inducible NF-kappaB luciferase reporter Measured in Cell-Based System Using Plate Reader - 2122-01_Inhibitor_SinglePoint_HTS_Activity

Keywords: NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2 ..more
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 Tested Compounds
 Tested Compounds
All(345051)
 
 
Active(2373)
 
 
Inactive(342421)
 
 
Inconclusive(266)
 
 
 Tested Substances
 Tested Substances
All(345298)
 
 
Active(2374)
 
 
Inactive(342658)
 
 
Inconclusive(266)
 
 
AID: 504558
Data Source: Broad Institute (2122-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-24

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2373
Related Experiments
Show more
AIDNameTypeComment
504586Broad Institute Epstein-Barr LMP-1 Inhibitor Probe ProjectSummarydepositor-specified cross reference
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
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

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 primary screen uses a stably transfected HEK293 cell line with a doxycycline & 4-hydroxytamoxifen inducible LMP1 TES2 construct and a NF-kappaB luciferase reporter. Induction of the system will activate the NF-kappaB pathway and result in expression of the luciferase reporter gene. Inhibitors of the the pathway will block expression of the reporter construct.

Expected Outcome: Inhibitors of the LMP1 pathway will show a reduction in luminescence measured by a commercial luciferase kit (SteadyGlo, Promega.) Later assays will determine whether this loss of signal is due to inhibition of the pathway of interest or due to off-target or general toxicity effects.
Protocol
LMP-1 Screening Protocol
(Luciferase reporter assay)

Day 0, cell grown in HyperFlask (Corning) 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 3000 per well in 20 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 30 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (HiRes Biosolutions). Final compound library concentration was 12.5 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 16.7 uM.
Incubate 16 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.

Day 3, remove plate from incubator to cool for 15 minutes to room temperature; add 20 uL 50% Promega Steady glo (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) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to -1 * the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 40.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid
Float
2REPLICATE_A_ACTIVITY_SCORE_12.5uM_(%) (12.5μM**)The calculated activity for the indicated sampleFloat%
3REPLICATE_B_ACTIVITY_SCORE_12.5uM_(%) (12.5μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_C_ACTIVITY_SCORE_12.5uM_(%) (12.5μM**)The calculated activity for the indicated sampleFloat%
5REPLICATE_D_ACTIVITY_SCORE_12.5uM_(%) (12.5μM**)The calculated activity for the indicated sampleFloat%

** 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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