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BioAssay: AID 489006

Dose Response selectivity of uHTS chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation in Jurkat cells using a luminescence assay

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(469)
 
 
Active(148)
 
 
Inactive(321)
 
 
 Tested Substances
 Tested Substances
All(470)
 
 
Active(148)
 
 
Inactive(322)
 
 
 Related BioAssays
 Related BioAssays
AID: 489006
Data Source: Burnham Center for Chemical Genomics (SBCCG-A521-NFkB-Jurkat-DR-B-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-11-15
Modify Date: 2010-12-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 148
Related Experiments
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AIDNameTypeProbeComment
465Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activationScreening depositor-specified cross reference
435022uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationScreening depositor-specified cross reference
435025Summary assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationSummary2 depositor-specified cross reference
449746Single concentration confirmation of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationScreening same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
493251SAR analysis of small molecule cytotoxicity in HEK293 cells for activators of NF-kappaB using a luminescence assayConfirmatory same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 T cell lines. Therefore, in this rescreening paradigm, the primary screen will be accomplished using an NF-kB-luciferase reporter cloned into a T-cell line (Jurkat). 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 T-cell specific manner.

The purpose of this assay is as a counterscreen for "uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation"(AID 435022).

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) Jurkat NF-kB 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
Day 1 Procedure
1) Harvest Jurkat NF-kB-Luc at 1000,000 cells/mL density. Spin, resuspend in assay media, count.
2) Seed 8000 cells/well in 3 uL/well to full plate in white 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) With a LabCyte Echo, dispense varying volumes of 10 mM test compounds in DMSO to achieve dose response concentrations and range (col 5-48) and backfill to equilibrate DMSO concentrations. To control wells (col 1-4), transfer equal volume of DMSO.
6) Incubate plate for 1 hour in 37oC 5% CO2.
7) Add 2uL PMA/Ionomycin working dilution (7.5 ng/mL PMA/3.75 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 37oC 5% CO2 incubator.
Day 2 Procedure
1) Remove lid and incubate plate for 10 min in at room temp.
2) Add 3ul/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 with and IC50_Mean <= 20 uM 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 and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: Jurkat
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 80 uM_first_point (80μM**)% Inhibition at a given concentrationFloat%
9% Activity at 40 uM_first_point (40μM**)% Inhibition at a given concentrationFloat%
10% Activity at 20 uM_first_point (20μM**)% Inhibition at a given concentrationFloat%
11% Activity at 10 uM_first_point (10μM**)% Inhibition at a given concentrationFloat%
12% Activity at 5 uM_first_point (5μM**)% Inhibition at a given concentrationFloat%
13% Activity at 2.5 uM_first_point (2.5μM**)% Inhibition at a given concentrationFloat%
14% Activity at 1.25 uM_first_point (1.25μM**)% Inhibition at a given concentrationFloat%
15% Activity at 0.625 uM_first_point (0.625μM**)% Inhibition at a given concentrationFloat%
16% Activity at 0.3125 uM_first_point (0.3125μM**)% Inhibition at a given concentrationFloat%
17% Activity at 0.15625 uM_first_point (0.15625μM**)% Inhibition at a given concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 80 uM_second_point (80μM**)% Inhibition at a given concentrationFloat%
20% Activity at 40 uM_second_point (40μM**)% Inhibition at a given concentrationFloat%
21% Activity at 20 uM_second_point (20μM**)% Inhibition at a given concentrationFloat%
22% Activity at 10 uM_second_point (10μM**)% Inhibition at a given concentrationFloat%
23% Activity at 5 uM_second_point (5μM**)% Inhibition at a given concentrationFloat%
24% Activity at 2.5 uM_second_point (2.5μM**)% Inhibition at a given concentrationFloat%
25% Activity at 1.25 uM_second_point (1.25μM**)% Inhibition at a given concentrationFloat%
26% Activity at 0.625 uM_second_point (0.625μM**)% Inhibition at a given concentrationFloat%
27% Activity at 0.3125 uM_second_point (0.3125μM**)% Inhibition at a given concentrationFloat%
28% Activity at 0.15625 uM_second_point (0.15625μM**)% Inhibition at a given concentrationFloat%
29IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2IC50 value determined using sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2Standard Error of IC50 valueFloatμM
32nH_2Hill coefficient determined using sigmoidal dose response equationFloat
33Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% Activity at 80 uM_third_point (80μM**)% Inhibition at a given concentrationFloat%
35% Activity at 40 uM_third_point (40μM**)% Inhibition at a given concentrationFloat%
36% Activity at 20 uM_third_point (20μM**)% Inhibition at a given concentrationFloat%
37% Activity at 10 uM_third_point (10μM**)% Inhibition at a given concentrationFloat%
38% Activity at 5 uM_third_point (5μM**)% Inhibition at a given concentrationFloat%
39% Activity at 2.5 uM_third_point (2.5μM**)% Inhibition at a given concentrationFloat%
40% Activity at 1.25 uM_third_point (1.25μM**)% Inhibition at a given concentrationFloat%
41% Activity at 0.625 uM_third_point (0.625μM**)% Inhibition at a given concentrationFloat%
42% Activity at 0.3125 uM_third_point (0.3125μM**)% Inhibition at a given concentrationFloat%
43% Activity at 0.15625 uM_third_point (0.15625μM**)% Inhibition at a given concentrationFloat%
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 80 uM_fourth_point (80μM**)% Inhibition at a given concentrationFloat%
46% Activity at 40 uM_fourth_point (40μM**)% Inhibition at a given concentrationFloat%
47% Activity at 20 uM_fourth_point (20μM**)% Inhibition at a given concentrationFloat%
48% Activity at 10 uM_fourth_point (10μM**)% Inhibition at a given concentrationFloat%
49% Activity at 5 uM_fourth_point (5μM**)% Inhibition at a given concentrationFloat%
50% Activity at 2.5 uM_fourth_point (2.5μM**)% Inhibition at a given concentrationFloat%
51% Activity at 1.25 uM_fourth_point (1.25μM**)% Inhibition at a given concentrationFloat%
52% Activity at 0.625 uM_fourth_point (0.625μM**)% Inhibition at a given concentrationFloat%
53% Activity at 0.3125 uM_fourth_point (0.3125μM**)% Inhibition at a given concentrationFloat%
54% Activity at 0.15625 uM_fourth_point (0.15625μM**)% Inhibition at a given concentrationFloat%

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

Data Table (Concise)
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