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BioAssay: AID 465

Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation

NF-kB, NF-kappaB, transcription factor, antigen receptor, Protein Kinase C-theta, PMA, ionomycin, luciferase, luminescence ..more
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 Tested Compounds
 Tested Compounds
All(61605)
 
 
Active(128)
 
 
Inactive(61477)
 
 
 Tested Substances
 Tested Substances
All(61609)
 
 
Active(128)
 
 
Inactive(61481)
 
 
 Related BioAssays
 Related BioAssays
AID: 465
Data Source: The Scripps Research Institute Molecular Screening Center ((8.1) NFkB_INH_Lumi_1536_%INH_Primary)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-09-25
Modify Date: 2009-03-26

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 128
Related Experiments
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AIDNameTypeComment
586Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activationConfirmatorydepositor-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
1266PMA/Ionomycin induced NF-kB luciferase assay in 293- NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1269In vitro luciferase assay using purified luciferase.Confirmatorydepositor-specified cross reference
1270PMA/Ionomycin-induced IL-8 production in HEK293- NF-kB-Luc stable cells.Confirmatorydepositor-specified cross reference
1271IL-2 Production Assay in Jurkat T-cells Stimulated with anti-CD3/anti-CD28 antibodiesConfirmatorydepositor-specified cross reference
1280In Vitro Kinase Assay Using Purified Enzyme PKC-betaConfirmatorydepositor-specified cross reference
1281PDBu Induced IL-8 Production in HEK293-NF-kB-luc Stable Cells.Confirmatorydepositor-specified cross reference
1282In Vitro Kinase Assay Using Purified Enzyme IKK-betaConfirmatorydepositor-specified cross reference
1283In Vitro Kinase Assay Using Purified Enzyme PKC-thetaConfirmatorydepositor-specified cross reference
1287TNF Induced NF-kB Luciferase in 293-NF-kB-Luc Stable CellsConfirmatorydepositor-specified cross reference
1288Confirmatory Screen: Chemical Inhibitors of TNF alpha stimulated E Selectin expressionConfirmatorydepositor-specified cross reference
1289CD40 overexpression induced NF-kB luciferase in HEK293-NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1290NOD1 overexpression induced NF-kB luciferase in 293-NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1291Doxorubincin induced NF-kB luciferase in HEK 293-NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1292Retinoic acid induced NF-kB luciferase in HEK293-NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1293NOD2 overexpression induced NF-kB luciferase in 293-NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1294CD4-TLR4 overexpresssion induced NF-kB luciferase in 293-NF-kB-Luc stable cellsConfirmatorydepositor-specified cross reference
1295PMA/Ionomycin-stimulated NF-kB DNA-binding activityConfirmatorydepositor-specified cross reference
1299Cytotoxicity Assay in THP.1 Cells.Confirmatorydepositor-specified cross reference
1302Cytotoxicity Assay in HeLa Cells.Confirmatorydepositor-specified cross reference
1364Anti-cd3/cd28 induced T cell proliferation of mouse primary lymphocytesConfirmatorydepositor-specified cross reference
1365Anti-IgM induced B cell proliferation of mouse primary lymphocytesConfirmatorydepositor-specified cross reference
1366PMA/Ionomycin induced NF-kB luciferase in MCF7 cellsConfirmatorydepositor-specified cross reference
1367Cytotoxicity assay in Jurkat T cells.Confirmatorydepositor-specified cross reference
1368Cytotoxicity assay in HEK293 cells.Confirmatorydepositor-specified cross reference
1369LPS induced NF-kB in THP.1 cellsConfirmatorydepositor-specified cross reference
1370GM-Tri-DAP induced IL-8 production in MCF-7/NOD1 cellsConfirmatorydepositor-specified cross reference
1371PMA/Ionomycin induced NF-kB luciferase in HEK293T cellsConfirmatorydepositor-specified cross reference
1372Cytotoxicity assay in HEK293T cells.Confirmatorydepositor-specified cross reference
1373Cytotoxicity assay in MCF7 cells.Confirmatorydepositor-specified cross reference
1374PMA/Ionomycin induced NF-kB luciferase in HeLa cellsConfirmatorydepositor-specified cross reference
1375PMA/Ionomycin induced NF-kB luciferase in PPC-1 cellsConfirmatorydepositor-specified cross reference
1384Chemical inhibitors of antigen receptor-induced NF-kB activationSummarydepositor-specified cross reference
1664Summary of the probe development efforts to identify chemical inhibitors of antigen receptor-induced NF-κB activationSummarydepositor-specified cross reference
435003uHTS luminescence assay for the identification of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-specified cross reference
435020Single concentration confirmation of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-specified cross reference
435022uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-specified cross reference
449746Single concentration confirmation of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-specified cross reference
489004Dose response confirmation of uHTS of chemical inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489006Dose Response selectivity of uHTS chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation in Jurkat cells using a luminescence assayConfirmatorydepositor-specified cross reference
489019Dose response cytoxicity of uHTS chemical inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489020Dose response counterscreen of uHTS chemical inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489021Dose response cytotoxicity of uHTS chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489023Dose response confirmation of uHTS of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489033Dose Response selectivity of uHTS chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489034Dose response confirmation of uHTS chemical inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489035Dose response confirmation of uHTS chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504485SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504487SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504489SAR Analysis of small molecule inhibitors of B-cell and T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504506SAR Analysis of small molecule inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504508SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
504512SAR Analysis of small molecule inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504514SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504515SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
504516SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504528SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation cytoxicity in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504641SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assay - Set 3Confirmatorydepositor-specified cross reference
504664SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
504665SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
504673SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assay - Set 3Confirmatorydepositor-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)
Grant Proposal number: 1 X01 MH077633-01

Keywords:
NF-kB, NF-kappaB, transcription factor, antigen receptor, Protein Kinase C-theta, PMA, ionomycin, luciferase, luminescence

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 NF-kB activation pathway.

[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
Protocol
Assay Overview:

This 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 NFkB response elements. As previously described, stimulation with a PMA/Ionomycin mix results in NF-kB activation, thus increasing luciferase expression and luminescence levels 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 primary HTS assay was conducted in 1536-well format. All compounds were tested once at a 3 micromolar final concentration.
A mathematical algorithm was used to determine nominally active compounds.
Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater %inhibition than the cutoff parameter was declared active.

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 (Kalypsys part# K1536 SWST)
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 compounds (3 micromolar, final DMSO concentration of 0.30%). 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 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 (30s per plate) with the ViewLux reader (Perkin Elmer).

The percent inhibition of each compound has been calculated as follow:

%inhibition = (1-(test_compound - Median_positive_control)/(Median_positive_control - Median_negative_control)*100

with positive control : 10 micromolar of the reference inhibitor #5653914 (ChemBridge)
and negative control : DMSO only

All data reported were normalized on a per-plate basis.
Comment
Possible artifacts of this assay can include, but are not limited to:
toxic compounds, compounds that inhibit luciferase activity, compounds that non-specifically inhibit transcriptional activity.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition_PrimaryNormalized percent inhibition of the primary screen at a compound concentration of 3 micromolarFloat%

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