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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 ):           Active    All
BioActive Compounds: 128
Depositor Specified Assays
Show more
AIDNameTypeComment
586Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activationconfirmatory
802Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expressionscreening
808Primary HTS assay for chemical potentiators of TNFalpha stimulated VCAM1 expressionscreening
819Primary HTS assay for chemical potentiators of IL-1B stimulated NFkB nuclear translocationscreening
836Primary HTS assay for chemical modifiers of cytoskeleton assemblyscreening
1013Confirmatory Screen for chemical inhibitors of TNF alpha stimulated VCAM1 expressionconfirmatory
1034Confirmatory Screen for chemical potentiatiors of TNF alpha stimulated VCAM1 expressionother
1246Primary HTS assay for chemical inhibitors of TNF alpha stimulated E-Selectin expressionscreening
1249Confirmatory Screen for chemical modifiers of cytoskeleton assemblyconfirmatory
1266PMA/Ionomycin induced NF-kB luciferase assay in 293- NF-kB-Luc stable cellsconfirmatory
1269In vitro luciferase assay using purified luciferase.confirmatory
1270PMA/Ionomycin-induced IL-8 production in HEK293- NF-kB-Luc stable cells.confirmatory
1271IL-2 Production Assay in Jurkat T-cells Stimulated with anti-CD3/anti-CD28 antibodiesconfirmatory
1280In Vitro Kinase Assay Using Purified Enzyme PKC-betaconfirmatory
1281PDBu Induced IL-8 Production in HEK293-NF-kB-luc Stable Cells.confirmatory
1282In Vitro Kinase Assay Using Purified Enzyme IKK-betaconfirmatory
1283In Vitro Kinase Assay Using Purified Enzyme PKC-thetaconfirmatory
1287TNF Induced NF-kB Luciferase in 293-NF-kB-Luc Stable Cellsconfirmatory
1288Confirmatory Screen: Chemical Inhibitors of TNF alpha stimulated E Selectin expressionconfirmatory
1289CD40 overexpression induced NF-kB luciferase in HEK293-NF-kB-Luc stable cellsconfirmatory
1290NOD1 overexpression induced NF-kB luciferase in 293-NF-kB-Luc stable cellsconfirmatory
1291Doxorubincin induced NF-kB luciferase in HEK 293-NF-kB-Luc stable cellsconfirmatory
1292Retinoic acid induced NF-kB luciferase in HEK293-NF-kB-Luc stable cellsconfirmatory
1293NOD2 overexpression induced NF-kB luciferase in 293-NF-kB-Luc stable cellsconfirmatory
1294CD4-TLR4 overexpresssion induced NF-kB luciferase in 293-NF-kB-Luc stable cellsconfirmatory
1295PMA/Ionomycin-stimulated NF-kB DNA-binding activityconfirmatory
1299Cytotoxicity Assay in THP.1 Cells.confirmatory
1302Cytotoxicity Assay in HeLa Cells.confirmatory
1364Anti-cd3/cd28 induced T cell proliferation of mouse primary lymphocytesconfirmatory
1365Anti-IgM induced B cell proliferation of mouse primary lymphocytesconfirmatory
1366PMA/Ionomycin induced NF-kB luciferase in MCF7 cellsconfirmatory
1367Cytotoxicity assay in Jurkat T cells.confirmatory
1368Cytotoxicity assay in HEK293 cells.confirmatory
1369LPS induced NF-kB in THP.1 cellsconfirmatory
1370GM-Tri-DAP induced IL-8 production in MCF-7/NOD1 cellsconfirmatory
1371PMA/Ionomycin induced NF-kB luciferase in HEK293T cellsconfirmatory
1372Cytotoxicity assay in HEK293T cells.confirmatory
1373Cytotoxicity assay in MCF7 cells.confirmatory
1374PMA/Ionomycin induced NF-kB luciferase in HeLa cellsconfirmatory
1375PMA/Ionomycin induced NF-kB luciferase in PPC-1 cellsconfirmatory
1384Chemical inhibitors of antigen receptor-induced NF-kB activationsummary
1664Summary of the probe development efforts to identify chemical inhibitors of antigen receptor-induced NF-κB activationsummary
435003uHTS luminescence assay for the identification of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activationscreening
435020Single concentration confirmation of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activationscreening
435022uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationscreening
449746Single concentration confirmation of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationscreening
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 assayconfirmatory
489006Dose Response selectivity of uHTS chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation in Jurkat cells using a luminescence assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
504485SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayconfirmatory
504487SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayconfirmatory
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 assayconfirmatory
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 assayconfirmatory
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 2confirmatory
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 assayconfirmatory
504514SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayconfirmatory
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 2confirmatory
504516SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayconfirmatory
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 assayconfirmatory
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 3confirmatory
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 2confirmatory
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 2confirmatory
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 3confirmatory
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)
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