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BioAssay: AID 504506

SAR 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 assay

Multiple cellular stimuli acting through various pathways lead to NF-kB induction. The assay described below uses tumor necrosis factor alpha (TNFa), a canonical NF-kB inducer, and is designed for identification of hits specific to TNFa-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the NF-kB primary screening of the library in B- and T-Cell- specific assays (AIDs 435022 and 435003). The HEK-293-T NF-kB-Luc cell line designed for luminescent detection of NF-kB induction was utilized in this assay. ..more
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AID: 504506
Data Source: Burnham Center for Chemical Genomics (SBCCG-A607-NFkB-TNFa-DryPowder-BT-Counterscreen-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-03-21
Hold-until Date: 2011-09-30
Modify Date: 2011-10-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
465Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activationScreeningdepositor-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
435022uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-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
489029Summary assay for inhibitors of both B-cell and T-cell specific antigen receptor-induced NF-kB activationSummarydepositor-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 assayConfirmatorysame project related to Summary assay
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 assayConfirmatorysame project related to Summary assay
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 assayConfirmatorysame project related to Summary assay
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 assayConfirmatorysame project related to Summary assay
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 assayConfirmatorysame 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

Multiple cellular stimuli acting through various pathways lead to NF-kB induction. The assay described below uses tumor necrosis factor alpha (TNFa), a canonical NF-kB inducer, and is designed for identification of hits specific to TNFa-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the NF-kB primary screening of the library in B- and T-Cell- specific assays (AIDs 435022 and 435003). The HEK-293-T NF-kB-Luc cell line designed for luminescent detection of NF-kB induction was utilized in this assay.

This dose response assay is developed and performed to counterscreen hits originally identified in "uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation" (AID 435022) and "uHTS luminescence assay for the identification of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation" (AID 435003) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

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) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory
2) Assay Media: DMEM with no phenol red (Gibco 31053 or Hyclone SH30585.02), 2mM L-glutamine (omega sci GS-60 200mM), 1mM Na-pyruvate (Sigma S8636-100ML 100mM), 50IU/ml Penn/Strep(omega scientific PS-20 5000 IU/mL)
3) Recombinant Human TNF-alpha, CF , (R&D Systems 210-TA-010/CF)
4) SteadyGlo (Promega E2550)
5) Assay plate: 1536-well, TC-treated, black plate (Corning 3893)
TNF-a NF-kB induction assay: Dose Response
Day 1 Procedure
1) Harvest HEK-293-T NF-kB-Luc at 100% confluency
2) Make cells suspension at 1X for final seeding density of 6000 cells/well in 5uL well volume.
3) Add TNF-a to 1X cell suspension at 0.1 ng/mL.
4) Dispense 6000 cells/well in 5 uL of assay media into col. 5-48 of assay plate
5) Dispense 5 uL/well of assay media into col. 1-4 of assay plate
6) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge
7) With LabCyte Echo, dispense varying volumes of test compounds in DMSO into assay plate wells (Col. 5-44) to achieve appropriate dose response concentrations and range. Backfill test compound wells with DMSO to equilibrate DMSO concentrations. Transfer equal volumes of DMSO to control wells (Col. 1-4, 45-48). Maximum concentration of DMSO should be 1%.
8) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O
9) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).
10) Incubate overnight (14 hours) in 37 oC 5% CO2 incubator
Day 2 Procedure
1) Add 3 ul/well SteadyGlo to each well with Multidrop.
2) Spin plates at 1000 rpm for 1 minute on Eppendorf 5810 centrifuge
3) Read luminescence on Perkin-Elmer EnVision
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 and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound 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-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 this 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 the items discussed above is
Score = 82 + 3*(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 85 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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: HEK293
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 the 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 a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1_Range1This qualifier is to be used with the next TID, IC50_1. If the 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_1_Range1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1_Range1Standard Error of the IC50 valueFloatμM
6nH_1_Range1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_point_Range1Flags 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 79 uM_first_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
9% Activity at 39.5 uM_first_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
10% Activity at 19.75 uM_first_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
11% Activity at 9.875 uM_first_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
12% Activity at 4.9375 uM_first_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
13% Activity at 2.46875 uM_first_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
14% Activity at 1.234375 uM_first_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
15% Activity at 0.6171875 uM_first_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
16% Activity at 0.3085938 uM_first_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
17% Activity at 0.1542969 uM_first_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
18Excluded_Points_second_point_Range1Flags 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 79 uM_second_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
20% Activity at 39.5 uM_second_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
21% Activity at 19.75 uM_second_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
22% Activity at 9.875 uM_second_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
23% Activity at 4.9375 uM_second_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
24% Activity at 2.46875 uM_second_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
25% Activity at 1.234375 uM_second_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
26% Activity at 0.6171875 uM_second_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
27% Activity at 0.3085938 uM_second_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
28% Activity at 0.1542969 uM_second_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
29IC50_Qualifier_2_Range1This qualifier is to be used with the next TID, IC50_2. If the 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_2_Range1IC50 value determined using a sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2_Range1Standard Error of the IC50 valueFloatμM
32nH_2_Range1Hill coefficient determined using sigmoidal dose response equationFloat
33Excluded_Points_third_point_Range1Flags 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 79 uM_third_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
35% Activity at 39.5 uM_third_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
36% Activity at 19.75 uM_third_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
37% Activity at 9.875 uM_third_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
38% Activity at 4.9375 uM_third_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
39% Activity at 2.46875 uM_third_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
40% Activity at 1.234375 uM_third_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
41% Activity at 0.6171875 uM_third_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
42% Activity at 0.3085938 uM_third_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
43% Activity at 0.1542969 uM_third_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
44Excluded_Points_fourth_point_Range1Flags 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 79 uM_fourth_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
46% Activity at 39.5 uM_fourth_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
47% Activity at 19.75 uM_fourth_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
48% Activity at 9.875 uM_fourth_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
49% Activity at 4.9375 uM_fourth_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
50% Activity at 2.46875 uM_fourth_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
51% Activity at 1.234375 uM_fourth_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
52% Activity at 0.6171875 uM_fourth_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
53% Activity at 0.3085938 uM_fourth_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
54% Activity at 0.1542969 uM_fourth_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
55IC50_Qualifier_3_Range1This qualifier is to be used with the next TID, IC50_3. If the 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
56IC50_3_Range1IC50 value determined using a sigmoidal dose response equationFloatμM
57Std.Err(IC50)_3_Range1Standard Error of the IC50 valueFloatμM
58nH_3_Range1Hill coefficient determined using sigmoidal dose response equationFloat
59Excluded_Points_fifth_point_Range1Flags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
60% Activity at 79 uM_fifth_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
61% Activity at 39.5 uM_fifth_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
62% Activity at 19.75 uM_fifth_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
63% Activity at 9.875 uM_fifth_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
64% Activity at 4.9375 uM_fifth_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
65% Activity at 2.46875 uM_fifth_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
66% Activity at 1.234375 uM_fifth_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
67% Activity at 0.6171875 uM_fifth_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
68% Activity at 0.3085938 uM_fifth_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
69% Activity at 0.1542969 uM_fifth_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
70Excluded_Points_sixth_point_Range1Flags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
71% Activity at 79 uM_sixth_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
72% Activity at 39.5 uM_sixth_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
73% Activity at 19.75 uM_sixth_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
74% Activity at 9.875 uM_sixth_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
75% Activity at 4.9375 uM_sixth_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
76% Activity at 2.46875 uM_sixth_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
77% Activity at 1.234375 uM_sixth_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
78% Activity at 0.6171875 uM_sixth_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
79% Activity at 0.3085938 uM_sixth_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
80% Activity at 0.1542969 uM_sixth_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
81IC50_Qualifier_4_Range1This qualifier is to be used with the next TID, IC50_4. If the 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
82IC50_4_Range1IC50 value determined using a sigmoidal dose response equationFloatμM
83Std.Err(IC50)_4_Range1Standard Error of the IC50 valueFloatμM
84nH_4_Range1Hill coefficient determined using sigmoidal dose response equationFloat
85Excluded_Points_seventh_point_Range1Flags to indicate which of the seventh dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
86% Activity at 79 uM_seventh_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
87% Activity at 39.5 uM_seventh_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
88% Activity at 19.75 uM_seventh_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
89% Activity at 9.875 uM_seventh_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
90% Activity at 4.9375 uM_seventh_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
91% Activity at 2.46875 uM_seventh_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
92% Activity at 1.234375 uM_seventh_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
93% Activity at 0.6171875 uM_seventh_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
94% Activity at 0.3085938 uM_seventh_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
95% Activity at 0.1542969 uM_seventh_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%
96Excluded_Points_eighth_point_Range1Flags to indicate which of the eighth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
97% Activity at 79 uM_eighth_point_Range1 (79μM**)% inhibition at the test concentrationFloat%
98% Activity at 39.5 uM_eighth_point_Range1 (39.5μM**)% inhibition at the test concentrationFloat%
99% Activity at 19.75 uM_eighth_point_Range1 (19.75μM**)% inhibition at the test concentrationFloat%
100% Activity at 9.875 uM_eighth_point_Range1 (9.875μM**)% inhibition at the test concentrationFloat%
101% Activity at 4.9375 uM_eighth_point_Range1 (4.9375μM**)% inhibition at the test concentrationFloat%
102% Activity at 2.46875 uM_eighth_point_Range1 (2.46875μM**)% inhibition at the test concentrationFloat%
103% Activity at 1.234375 uM_eighth_point_Range1 (1.23438μM**)% inhibition at the test concentrationFloat%
104% Activity at 0.6171875 uM_eighth_point_Range1 (0.617188μM**)% inhibition at the test concentrationFloat%
105% Activity at 0.3085938 uM_eighth_point_Range1 (0.308594μM**)% inhibition at the test concentrationFloat%
106% Activity at 0.1542969 uM_eighth_point_Range1 (0.154297μM**)% inhibition at the test concentrationFloat%

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

Data Table (Concise)
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