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BioAssay: AID 2793

SAR analysis of NF-kappaB dependent luciferase using DAP as an inducer - Set 2

The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis. ..more
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 Tested Compounds
 Tested Compounds
All(16)
 
 
Active(3)
 
 
Inactive(13)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Active(3)
 
 
Inactive(13)
 
 
AID: 2793
Data Source: Burnham Center for Chemical Genomics (BCCG-A342-NOD1-DAP-DryPowder-Assay-2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-04-28
Hold-until Date: 2010-09-02
Modify Date: 2011-01-06

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeProbeComment
1566uHTS luminescence assay for the identification of compounds that inhibit NOD2Confirmatory depositor-specified cross reference
1575Summary assay for the identification of compounds that inhibit NOD1Summary2 depositor-specified cross reference: Summary AID.
1578uHTS luminescence assay for the identification of compounds that inhibit NOD1Confirmatory depositor-specified cross reference
1579Summary assay for the identification of compounds that inhibit NOD2Summary depositor-specified cross reference: Summary AID.
1849uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assayConfirmatory depositor-specified cross reference
1852HTS assay for identification of inhibitors of TNF-a-specific NF-kB inductionOther depositor-specified cross reference
2264SAR analysis of NF-kB dependent luciferase using DAP as an inducerConfirmatory depositor-specified cross reference
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
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
2337SAR analysis of inhibitors of TNFa specific NF-kB induction 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
2485HTS dose response assay for identification of inhibitors of TNFa-specific NF-kB inductionConfirmatory 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
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
2260SAR analysis of muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells.Confirmatory 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
2337SAR analysis of inhibitors of TNFa specific NF-kB induction revisedConfirmatory same project related to Summary assay
2475SAR analysis of compounds that inhibit NOD2 - Set 2Confirmatory same project related to Summary assay
2485HTS dose response assay for identification of inhibitors of TNFa-specific NF-kB inductionConfirmatory 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
2799SAR analysis of compounds that inhibit NOD2 - 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 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA

The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis.

Mutations in the NOD1 and NOD2 genes are associated with a number of human inflammatory disorders, including Crohn's disease (CD), Blau syndrome, early-onset sarcoidosis, and atopic diseases, which characteristically cause constitutive NF-kB activation. Chemical inhibitors of NOD1 and NOD2 would provide powerful research tools for elucidating the roles of these proteins in primary cultured cells from humans and in animal models.

The assay described below is a cell-based confirmatory assay that utilizes NF-kB-mediated luciferase reporter gene activity as a measure of NOD1 modulation. The assay uses a luminescent readout.

This dose response assay is developed and performed to confirm hits originally identified in "uHTS luminescence assay for the identification of compounds that inhibit NOD1" (AID 1578) 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) Strober W, Murray PJ, Kitani A, Watanabe T. Nat Rev Immunol. 2006 Jan;6(1):9-20. Review. Signalling pathways and molecular interactions of NOD1 and NOD2.

2. da Silva Correia J, Miranda Y, Austin-Brown N, Hsu J, Mathison J, Xiang R, Zhou H, Li Q, Han J, Ulevitch RJ. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1840-5. Epub 2006 Jan 30. Nod1-dependent control of tumor growth

3. Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, Lebourhis L, Philpott DJ, Nahori MA, Popa C, Morre SA, van der Meer JW, Girardin SE, Netea MG, van den Berg WB. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9017-22. Epub 2008 Jun 23. Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis.

4. Shaw MH, Reimer T, Kim YG, Nunez G. Curr Opin Immunol. 2008 Aug;20(4):377-82. Epub 2008 Jul 2. Review. NOD-like receptors (NLRs): bona fide intracellular microbial sensors.

5.Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. Immunity. 2008 Feb;28(2):246-57. Epub 2008 Feb 7. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands.

6. Rescigno M, Nieuwenhuis EE. Curr Opin Gastroenterol. 2007 Jan;23(1):21-6. Review. The role of altered microbial signaling via mutant NODs in intestinal inflammation.

7. Rosenstiel P, Hellmig S, Hampe J, Ott S, Till A, Fischbach W, Sahly H, Lucius R, Folsch UR, Philpott D, Schreiber S. Cell Microbiol. 2006 Jul;8(7):1188-98. Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection.

8. McGovern DP, Hysi P, Ahmad T, van Heel DA, Moffatt MF, Carey A, Cookson WO, Jewell DP. Hum Mol Genet. 2005 May 15;14(10):1245-50. Epub 2005 Mar 24. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease.

9. Opitz B, Puschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, Schumann RR, Suttorp N, Hippenstiel S. J Biol Chem. 2004 Aug 27;279(35):36426-32. Epub 2004 Jun 23. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae.

10. Le Bourhis L, Benko S, Girardin SE. Biochem Soc Trans. 2007 Dec;35(Pt 6):1479-84. Review. Nod1 and Nod2 in innate immunity and human inflammatory disorders.

11. Maeda S, Hsu LC, Liu H, Bankston LA, Iimura M, Kagnoff MF, Eckmann L, Karin M. Science. 2005 Feb 4;307(5710):734-8. Erratum in: Science. 2005 Apr 29;308(5722):633. Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing

12. Li J, Moran T, Swanson E, Julian C, Harris J, Bonen DK, Hedl M, Nicolae DL, Abraham C, Cho JH. Regulation of IL-8 and IL-1beta expression in Crohn's disease associated NOD2/CARD15 mutations.

13. Hum Mol Genet. 2004 Aug 15;13(16):1715-25. Epub 2004 Jun 15. Brideau C, Gunter B, Pikounis B, Liaw A. J Biomol Screen. 2003 Dec;8(6):634-47 Improved statistical methods for hit selection in high-throughput screening.
Protocol
Assay materials:
1) HEK-293-T NFKB-Luc cell line obtained from the assay provider's laboratory.
2) g-tri-DAP (Ana Spec cat #60774) obtained from assay provider's laboratory.
3) SteadyGlo (Promega)

Protocol:

1. Compounds were diluted in DMEM 10% FBS plus Pen/Strep (culture medium), and 10 uL were added into respective wells (96-well culture plate) to reach desired final concentrations.
2. G-tri-DAP was added, in a final concentration of 5,4 ug/ml, to a culture medium suspension of HEK-293-T NFKB-Luc cells (1,1x10+6 cells per mililiter).
3. Pre-induced HEK-293-T NFKB-Luc cells were added into the respective wells (90 ul per well) and kept at 37oC (10% CO2 incubator) for 18 hours.
4. After incubation, plated cells were equilibrated to room temperature for 15 minutes.
5.Luciferase assay was then performed by adding 40 microliters per well of the Steady-GloTM reagent. After a ten minute incubation at room temperature, light emission was measured with the AnalystTM Microplate reader (LJL Biosystems).
Comment
Compounds are considered active if < 25 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 to this assay

2) 2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to 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's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3),
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 Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. 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.String
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
5% Activity at 25_uM_point_1 (25μM**)% inhibition at the test concentrationFloat%
6% Activity at 25_uM_point_2 (25μM**)% inhibition at the test concentrationFloat%
7% Activity at 25_uM_point_3 (25μM**)% inhibition at the test concentrationFloat%
8% Activity at 10_uM_point_1 (10μM**)% inhibition at the test concentrationFloat%
9% Activity at 10_uM_point_2 (10μM**)% inhibition at the test concentrationFloat%
10% Activity at 10_uM_point_3 (10μM**)% inhibition at the test concentrationFloat%
11% Activity at 5_uM_point_1 (5μM**)% inhibition at the test concentrationFloat%
12% Activity at 5_uM_point_2 (5μM**)% inhibition at the test concentrationFloat%
13% Activity at 5_uM_point_3 (5μM**)% inhibition at the test concentrationFloat%
14% Activity at 2.5_uM_point_1 (2.5μM**)% inhibition at the test concentrationFloat%
15% Activity at 2.5_uM_point_2 (2.5μM**)% inhibition at the test concentrationFloat%
16% Activity at 2.5_uM_point_3 (2.5μM**)% inhibition at the test concentrationFloat%
17% Activity at 1_uM_point_1 (1μM**)% inhibition at the test concentrationFloat%
18% Activity at 1_uM_point_2 (1μM**)% inhibition at the test concentrationFloat%
19% Activity at 1_uM_point_3 (1μM**)% inhibition at the test concentrationFloat%
20% Activity at 0.25_uM_point_1 (0.25μM**)% inhibition at the test concentrationFloat%
21% Activity at 0.25_uM_point_2 (0.25μM**)% inhibition at the test concentrationFloat%
22% Activity at 0.25_uM_point_3 (0.25μM**)% inhibition at the test concentrationFloat%

* Activity Concentration. ** Test 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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