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BioAssay: AID 586

Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation

Name: Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation ..more
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 Tested Compounds
 Tested Compounds
All(112)
 
 
Active(78)
 
 
Inactive(34)
 
 
 Tested Substances
 Tested Substances
All(112)
 
 
Active(78)
 
 
Inactive(34)
 
 
 Related BioAssays
 Related BioAssays
AID: 586
Data Source: The Scripps Research Institute Molecular Screening Center (NFkB_INH_LUMI_1536_IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-08
Modify Date: 2009-03-26

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 78
Related Experiments
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AIDNameTypeComment
465Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activationScreeningdepositor-specified cross reference
802Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expressionScreeningdepositor-specified cross reference
808Primary HTS assay for chemical potentiators of TNFalpha stimulated VCAM1 expressionScreeningdepositor-specified cross reference
819Primary HTS assay for chemical potentiators of IL-1B stimulated NFkB nuclear translocationScreeningdepositor-specified cross reference
836Primary HTS assay for chemical modifiers of cytoskeleton assemblyScreeningdepositor-specified cross reference
1013Confirmatory Screen for chemical inhibitors of TNF alpha stimulated VCAM1 expressionConfirmatorydepositor-specified cross reference
1034Confirmatory Screen for chemical potentiatiors of TNF alpha stimulated VCAM1 expressionOtherdepositor-specified cross reference
1246Primary HTS assay for chemical inhibitors of TNF alpha stimulated E-Selectin expressionScreeningdepositor-specified cross reference
1249Confirmatory Screen for chemical modifiers of cytoskeleton assemblyConfirmatorydepositor-specified cross reference
1288Confirmatory Screen: Chemical Inhibitors of TNF alpha stimulated E Selectin expressionConfirmatorydepositor-specified cross reference
1664Summary of the probe development efforts to identify chemical inhibitors of antigen receptor-induced NF-κB activationSummarydepositor-specified cross reference
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: The Burnham Institute
Network: Molecular Library Screening Center Network (MLSCN)
Proposal number 1X01-MH077633-01

External Assay ID: NFkB_INH_LUMI_1536_IC50

Name: Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation

Description:
Many cellular pathways leading to activation of NF-kB-family transcription factors have been identified to be participating in host-defense, immunity, inflammation, and cancer. Recently, a unique pathway activated by antigen receptors on T- and B-Lymphocytes has been revealed, involving a cascade of participating proteins that includes CARMA1 (BIMP3), Bcl-10, paracaspase (MALT1), TRAF6, and Ubc13. This pathway is initiated by Protein Kinase C-theta, which induces phosphorylation of components of this signaling pathway [1]. Based on experiments using siRNA and dominant-negative mutants, it has been determined that treatment of cells with the combination of phorbol ester PMA and the calcium-ionophore ionomycin triggers this pathway, resulting in NF-kB activation [2-5]. Compounds able to block this stimulation will be useful research tools for analysis of the physiological roles of this new NF-kB activation pathway.

References:
[1]Thome M. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat Rev Immunol. 2004 May;4(5):348-59. Review
[2]Ruland J, Duncan GS, Elia A, del Barco Barrantes I, Nguyen L, Plyte S, Millar DG, Bouchard D, Wakeham A, Ohashi PS, Mak TW. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure. Cell. 2001 Jan 12;104(1):33-42
[3]McAllister-Lucas LM, Inohara N, Lucas PC, Ruland J, Benito A, Li Q, Chen S, Chen FF, Yamaoka S, Verma IM, Mak TW, Nunez G. Bimp1, a
MAGUK family member linking protein kinase C activation to Bcl10-mediated NF-kappaB induction. J Biol Chem. 2001 Aug 17;276(33):30589-97
[4]Ruefli-Brasse AA, French DM, Dixit VM. Regulation of NF-kappaB-dependent lymphocyte activation and development by paracaspase. Science. 2003 Nov 28;302(5650):1581-4
[5]Zhou H, Wertz I, O'Rourke K, Ultsch M, Seshagiri S, Eby M, Xiao W, Dixit VM. Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO. Nature. 2004 Jan 8;427(6970):167-71

Keywords:
NF-kB, NF-kappaB, transcription factor, antigen receptor, Protein Kinase C-theta, PMA, ionomycin, luciferase, luminescence, Scripps
Protocol
Assay Overview:
Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID=465.
Among the 128 compounds identified during the primary screening, 112 were assessed in dose-response experiments in 10 point, 1:3 serial dilution starting at a nominal test concentration of 30 micromolar.
As with the primary screen, the assay utilizes HEK-293 cells that have been stably transfected with the mammalian expression plasmid
pUC13-4xNFkB-Luc, allowing the expression of the firefly luciferase under the control of four tandem HIV NF-kB response elements. Stimulation with a PMA/ionomycin mix results in NF-kB activation, thus increasing luciferase expression and luminescence levels as measured with an appropriate substrate. Compounds that decrease light emission are potential inhibitors and may be able to block the NF-kB activation triggered by the PMA/Ionomycin stimulation, hence providing valuable tools to decipher this new pathway.
The dose-response assay was conducted in 1536-well plate format.
Protocol Summary:
HEK-293 stably transfected with the pUC13-4xNFkB-Luc were cultured in T-175 flasks (Corning part#431080) at 37 degrees Celsius, 5%CO2 and 95% relative humidity. The growth media consisted of Dulbecco's Modified Eagle's Media (Invitrogen part# 11965-092) supplemented with 10% v/v heat inactivated fetal bovine serum (Hyclone part#SH30088), 1% v/v penicillin-streptomycin mix (Invitrogen part#15140-122) and 1ug/mL puromycin (InvivoGen part#ant-pr-1). Prior to the assay, cells were suspended to a concentration of 625,000 cells per milliliter in phenol red free Dulbecco's Modified Eagle's Media (Invitrogen part# 21063-029) supplemented with 10% v/v heat inactivated fetal bovine serum (Hyclone part#SH30088), 1% v/v penicillin-streptomycin mix (Invitrogen part#15140-122) and 1ug/mL puromycin (InvivoGen part#ant-pr-1).
The assay began by dispensing 4 microliters of cell suspension to each well (i.e. 2,500 cells/well) of a white solid-bottom 1536-well plate.
Plates were then placed in the incubator overnight at 37 degrees Celsius, 5%CO2 and 95% relative humidity. Twenty four hours after seeding, cells were treated with 15 nL/well of test compounds or positive and negative controls (10 micromolar of Chembridge compound part#5653914 and DMSO, respectively). Each compound dilution was assayed in triplicate, for a nominal total of 30 data points per dose response.
One hour later, cells were stimulated with 1 microliter per well of a PBS mix of PMA (100ng/mL, Calbiochem part#524400) and ionomycin (50ng/mL, Calbiochem part#407950). Plates were subsequently returned to the incubator for 16 hours (37 degrees Celsius, 5%CO2 and 95% relative humidity).
After the incubation, plates were equilibrated to room temperature for 20 minutes. A luciferase assay was then performed by adding 5 microliters per well of the SteadyLite HTS reagent (Perkin Elmer part#6016989).
After a 15 minutes incubation time, light emission was measured with the ViewLux reader (Perkin Elmer).
The percent inhibition of each well has been calculated as follow:
%inhibition = 100 * (1-(median_positive_control - test_compound)/(median_positive_control - median_negative_control))
with positive control : reference inhibitor #5653914 (ChemBridge, 10 micromolar)and negative control : DMSO only
For each compound, percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept.
In cases where the highest concentration tested (i.e. 30 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 30 micromolar. Compounds with IC50 of greater than 10 micromolar were considered inactive; compounds with IC50 of equal to or less than 10 micromolar were considered active.
Comment
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to:
toxic compounds, compounds that inhibit luciferase activity, compounds that non-specifically inhibit or activate transcriptional activity.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity QualifierActivity 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
2IC50Qualified IC50 in micromolar: The concentration at which 50% of the inhibition is observed (relative to 100% inhibition of 10 micromolar reference inhibitor)FloatμM
3LogIC50Log10 of the qualified IC50 in M concentration.Float
4Hill CoefficientThe 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
8Curve Chi2A 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
9Curve R2This value indicates 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
10Excluded PointsNumber of excluded point in the dose-response curve (counting one data point per concentration).Integer
11Number of DatapointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all

concentration points); in some cases a data point can be excluded as an outlier.
Integer
12Inhibition at 1.5 nMNormalized percent inhibition at 1.5 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 4.5 nMNormalized percent inhibition at 4.5 nanomolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 13.7 nMNormalized percent inhibition at 13.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 41 nMNormalized percent inhibition at 41 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 123 nMNormalized percent inhibition at 123 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 370 nMNormalized percent inhibition at 370 nanomolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 1.11 uMNormalized percent inhibition at 1.11 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 3.33 uMNormalized percent inhibition at 3.33 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 10 uMNormalized percent inhibition at 10 micromolar inhibitor concentration; average of triplicate measurement.Float%
21Inhibition at 30 uMNormalized percent inhibition at 30 micromolar inhibitor concentration; average of triplicate measurement.Float%

Data Table (Concise)
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