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BioAssay: AID 435022

uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation

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 more ..
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 Tested Compounds
 Tested Compounds
All(331675)
 
 
Active(1077)
 
 
Inactive(330601)
 
 
 Tested Substances
 Tested Substances
All(331759)
 
 
Active(1079)
 
 
Inactive(330680)
 
 
 Related BioAssays
 Related BioAssays
AID: 435022
Data Source: Burnham Center for Chemical Genomics (BCCG-A382-NFkB-697B-Primary-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-06-28
Modify Date: 2011-01-14

Data Table ( Complete ):           Active    All
BioActive Compounds: 1077
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
465Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activationscreening
435025Summary assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationsummary2
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
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
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
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
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
504670SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a HEK-293T 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
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
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
449746Single concentration confirmation of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationscreening
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
489022Dose response counterscreen 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
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
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
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
493251SAR analysis of small molecule cytotoxicity in HEK293 cells for activators of NF-kappaB using a luminescence assayconfirmatory
504518SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a HEK-293T 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
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
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
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
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 X01 MH077633-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA

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.

This assay was originally assigned to the Scripps Research Institute Molecular Screening Center (TSRI MSC) by the NIH during the MLSCN pilot phase (#Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation#, AID 465.) In the original MLSCN screen a stably transfected reporter epithelial cell line (HEK293) that contains a luciferase gene driven by a NF-kB responsive promoter was used for compound library screening. The hits identified from that screen were very much less potent when tested in B cell lines. Therefore, in this rescreening paradigm, the primary screen will be accomplished using an NF-kB-luciferase reporter cloned into a human derived B-cell line (697). The goal therefore is to identify compounds that selectively inhibit one of the several known pathways that lead to NF-kB activation in mammalian cells in a B-cell specific manner.


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
Protocol
Assay materials:
1) 697 NF-kB-Luc cell line obtained from Assay Provider
2) RPMI phenol red (-), Mediatech Car # 17105CV
3) Fetal Bovine Serum, Hyclone cat # SH30396.03HI
4) L-glutamine. Omega Scientific cat # GS-60
5) Na-pyruvate, Sigma cat # S8636-100M
6) Penn/Strep, Omega Scientific cat # PS-20
7) PMA, Calbiochem cat # 524400
8) Ionomycin, Calbiochem cat # 407950
9) SteadyGlo, Promega cat # E2550

Primary Screen and Single-concentration confirmation
Day 1 Procedure
1) Harvest 697 NF-kB-Luc at 2,000,000 cells/mL density. Spin, re-suspend in assay media, count.
2) Seed 15000 cells/well in 3 uL/well 697 NF-kB-Luc to full plate- Aurora 1536 low base white plate # 00029846 plate or non-TC-treated equivalent.
3) Plate with Kalypsys dispenser
4) Spin down plates @ 500 RPM for 1 min.
5) Dispense 10 nl 100% DMSO compounds (col 5-48) & 10nL 100% DMSO controls (col 1-4) to plates with Echo 555 (Labcyte) acoustic dispenser
6) Incubate plate for 1 hour in 37 oC 5% CO2.
7) Add 2uL PMA/Ionomycin working dilution (6.5 ng/mL PMA/3.25 ng/mL Ionomycin) in assay media to col 3-48 and no agonist media to col 1 and 2 with Kalypsys dispenser.
8) Spin down plates @ 1000 RPM for 1 min.
9) Lid plates with Kalypsys plate lids.
10) Incubate plate overnight (16 hours) in 37 oC 5% CO2 incubator.

Day 2 Procedure
1) Remove lid and incubate plate for 10 min in at room temp.
2) Add 3 ul/well SteadyGlo with Kalypsys dispenser
3) Spin plates @ 2000 RPM for 2 min, lid plate and incubate for 10 min at room temp.
4) Read luminescence on Envision Ultra sensitive Luminescent protocol for white Aurora 1536 low base white plate # 00029846
Comment
Compounds that demonstrated an inhibition of >= 35% at 4 uM concentration are defined as actives in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % inhibition in the assay demonstrated by a compound at 4 uM concentration:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 4 uM (4μM**)% inhibition in primary screeningFloat%
2Value (4μM**)Value of the sample at 4 uMFloatRLU
3Mean HighMean Fluorescence ratio of negative controls in the corresponding plateFloatRLU
4STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatRLU
5Mean LowMean Fluorescence ratio of positive controls in the corresponding plateFloatRLU
6STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatRLU

** Test Concentration.
Additional Information
Grant Number: 1 X01 MH077633-01

Data Table (Concise)
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