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BioAssay: AID 2485

HTS dose response assay for identification of inhibitors of TNFa-specific NF-kB induction

Multiple cellular stimuli acting through various pathways lead to NF-kB induction. The assay described below uses tumor necrosis factor alpha (TNF-a), a canonical NF-kB inducer, and is designed for identification of hits specific to TNF-a-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the primary screening of the library in NOD1- and NOD2-specific assays more ..
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 Tested Compounds
 Tested Compounds
All(546)
 
 
Active(437)
 
 
Inactive(109)
 
 
 Tested Substances
 Tested Substances
All(546)
 
 
Active(437)
 
 
Inactive(109)
 
 
AID: 2485
Data Source: Burnham Center for Chemical Genomics (BCCG-A305-TNF-Alpha-Inhibitor-DR-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-05
Modify Date: 2011-01-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 437
Related Experiments
Show more
AIDNameTypeProbeComment
1566uHTS luminescence assay for the identification of compounds that inhibit NOD2Confirmatory depositor-specified cross reference: uHTS luminescence assay for the identification of compounds that inhibit NOD2
1575Summary assay for the identification of compounds that inhibit NOD1Summary2 depositor-specified cross reference: Summary assay for the identification of compounds that inhibit NOD1
1578uHTS luminescence assay for the identification of compounds that inhibit NOD1Confirmatory depositor-specified cross reference: uHTS luminescence assay for the identification of compounds that inhibit NOD1
1579Summary assay for the identification of compounds that inhibit NOD2Summary depositor-specified cross reference: Summary assay for the identification of compounds that inhibit NOD2
1852HTS assay for identification of inhibitors of TNF-a-specific NF-kB inductionOther depositor-specified cross reference
2337SAR analysis of inhibitors of TNFa specific NF-kB induction revisedConfirmatory depositor-specified cross reference
1666SAR assay for compounds that inhibit PHOSPHO1Confirmatory same project related to Summary assay
1849uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assayConfirmatory same project related to Summary assay
2001uHTS luminescence assay for the identification of compounds that inhibit NOD2 in MDP treated cellsConfirmatory same project related to Summary assay
2245SAR analysis of tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells.Confirmatory same project related to Summary assay
2250SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cellsConfirmatory same project related to Summary assay
2255SAR analysis of NF-kB dependent luciferase using Doxorucibin as an inducerConfirmatory same project related to Summary assay
2260SAR analysis of muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells.Confirmatory same project related to Summary assay
2261SAR analysis of NF-kB dependent luciferase using PMA/Ionomycin as an inducerConfirmatory same project related to Summary assay
2264SAR analysis of NF-kB dependent luciferase using DAP as an inducerConfirmatory same project related to Summary assay
2333SAR analysis of compounds that inhibit NOD1 revisedConfirmatory same project related to Summary assay
2334SAR analysis of compounds that inhibit NOD2 revisedConfirmatory same project related to Summary assay
2335SAR analysis of compounds that are cytotoxic to HEK293 revisedConfirmatory same project related to Summary assay
2466SAR analysis of compounds that inhibit NOD1 - Set 2Confirmatory same project related to Summary assay
2469SAR analysis of compounds that are cytotoxic to HEK293 - Set 2Confirmatory same project related to Summary assay
2475SAR analysis of compounds that inhibit NOD2 - Set 2Confirmatory same project related to Summary assay
2483SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 2Confirmatory same project related to Summary assay
2503SAR analysis of Muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells - Set 2Confirmatory same project related to Summary assay
2504SAR analysis of Tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells - Set 2Confirmatory same project related to Summary assay
2505SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cells - Set 2Confirmatory same project related to Summary assay
2789SAR analysis of NF-kappaB dependent luciferase using Doxorucibin as an inducer - Set 2Confirmatory same project related to Summary assay
2792SAR analysis of NF-kappaB dependent luciferase using PMA/Ionomycin as an inducer - 2Confirmatory same project related to Summary assay
2793SAR analysis of NF-kappaB dependent luciferase using DAP as an inducer - Set 2Confirmatory same project related to Summary assay
2798SAR analysis of compounds that inhibit NOD1 - Set 3Confirmatory same project related to Summary assay
2799SAR analysis of compounds that inhibit NOD2 - Set 3Confirmatory same project related to Summary assay
2800SAR analysis of compounds that are cytotoxic to HEK293 - Set 3Confirmatory same project related to Summary assay
2801SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 3Confirmatory same project related to Summary assay
1848uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD2 cell inhibition assayConfirmatory same project related to Summary assay
2001uHTS luminescence assay for the identification of compounds that inhibit NOD2 in MDP treated cellsConfirmatory same project related to Summary assay
2245SAR analysis of tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells.Confirmatory same project related to Summary assay
2250SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cellsConfirmatory same project related to Summary assay
2255SAR analysis of NF-kB dependent luciferase using Doxorucibin as an inducerConfirmatory same project related to Summary assay
2260SAR analysis of muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells.Confirmatory same project related to Summary assay
2261SAR analysis of NF-kB dependent luciferase using PMA/Ionomycin as an inducerConfirmatory same project related to Summary assay
2264SAR analysis of NF-kB dependent luciferase using DAP as an inducerConfirmatory same project related to Summary assay
2333SAR analysis of compounds that inhibit NOD1 revisedConfirmatory same project related to Summary assay
2334SAR analysis of compounds that inhibit NOD2 revisedConfirmatory same project related to Summary assay
2335SAR analysis of compounds that are cytotoxic to HEK293 revisedConfirmatory same project related to Summary assay
2466SAR analysis of compounds that inhibit NOD1 - Set 2Confirmatory same project related to Summary assay
2469SAR analysis of compounds that are cytotoxic to HEK293 - Set 2Confirmatory same project related to Summary assay
2475SAR analysis of compounds that inhibit NOD2 - Set 2Confirmatory same project related to Summary assay
2483SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 2Confirmatory same project related to Summary assay
2503SAR analysis of Muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells - Set 2Confirmatory same project related to Summary assay
2504SAR analysis of Tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells - Set 2Confirmatory same project related to Summary assay
2505SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cells - Set 2Confirmatory same project related to Summary assay
2789SAR analysis of NF-kappaB dependent luciferase using Doxorucibin as an inducer - Set 2Confirmatory same project related to Summary assay
2792SAR analysis of NF-kappaB dependent luciferase using PMA/Ionomycin as an inducer - 2Confirmatory same project related to Summary assay
2793SAR analysis of NF-kappaB dependent luciferase using DAP as an inducer - Set 2Confirmatory same project related to Summary assay
2799SAR analysis of compounds that inhibit NOD2 - Set 3Confirmatory same project related to Summary assay
2800SAR analysis of compounds that are cytotoxic to HEK293 - Set 3Confirmatory same project related to Summary assay
2801SAR analysis of inhibitors of TNFa specific NF-kB induction - 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 Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084844-01
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 (TNF-a), a canonical NF-kB inducer, and is designed for identification of hits specific to TNF-a-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the primary screening of the library in NOD1- and NOD2-specific assays (AIDs 1578 and 1566). The HEK-293-T NF-kB-Luc cell line designed for luminescent detection of NF-kB induction was utilized in this assay. This assay is the dose response follow-up to "HTS assay for identification of inhibitors of TNF-a-specific NF-kB induction", AID 1852.
Protocol
Assay materials:
1) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory.
2) RNDsystems (Cat # 210-TA-010/CF)
3) SteadyGlo (Promega)
(This assay was multiplexed with a cytotoxicity protocol described in AID 1849).
Day 1 Procedure
1) Harvest HEK-293-T NFKB-Luc at 100% confluency
2) Add 1 uL/well NOD assay media with Multidrop
3) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.
4) Serial compound dilutions: dispense 50nl 100% DMSO (columns 1-4, 47-48) or compounds (columns 5-46) using with Labcyte Echo 550 into plates from step 2.
5) Add TNFa to cell suspension at 0.1 ng/mL.
6) Seed 6000 cells/well in 4 uL/well to full plate HEK-293-T NFKB-Luc to Corning # 3727 white, 1536, hi-profile, TC-treated plate.
7) Spin down plates @ 500 RPM for 5 min on Eppendorf 5810 centrifuge.
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 of BriteGlo solution with Multidrop
2) Shake plates on a plate shaker for 20 min.
3) Spin plates @ 1000 RPM for 1 min using Eppendorf 5810 centrifuge.
4) Read luminescence on Perkin-Elmer Viewlux.
Comment
Compounds are considered active if the IC50 < 20 uM.
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) 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 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 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 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: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. 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*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH084844-01

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