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BioAssay: AID 1578

uHTS luminescence assay for the identification of compounds that inhibit NOD1

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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AID: 1578
Data Source: Burnham Center for Chemical Genomics (BCCG-A153-NOD1-Primary-Luminescent-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-03-23
Modify Date: 2010-12-30

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 2997
Depositor Specified Assays
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AIDNameTypeProbeComment
1566uHTS luminescence assay for the identification of compounds that inhibit NOD2confirmatory
1849uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assayconfirmatory
1852HTS assay for identification of inhibitors of TNF-a-specific NF-kB inductionother
1575Summary assay for the identification of compounds that inhibit NOD1summary2
2333SAR analysis of compounds that inhibit NOD1 revisedconfirmatory
2250SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cellsconfirmatory
2255SAR analysis of NF-kB dependent luciferase using Doxorucibin as an inducerconfirmatory
2260SAR analysis of muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells.confirmatory
2261SAR analysis of NF-kB dependent luciferase using PMA/Ionomycin as an inducerconfirmatory
2264SAR analysis of NF-kB dependent luciferase using DAP as an inducerconfirmatory
2334SAR analysis of compounds that inhibit NOD2 revisedconfirmatory
2335SAR analysis of compounds that are cytotoxic to HEK293 revisedconfirmatory
2337SAR analysis of inhibitors of TNFa specific NF-kB induction revisedconfirmatory
2466SAR analysis of compounds that inhibit NOD1 - Set 2confirmatory
2469SAR analysis of compounds that are cytotoxic to HEK293 - Set 2confirmatory
2475SAR analysis of compounds that inhibit NOD2 - Set 2confirmatory
2483SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 2confirmatory
2485HTS dose response assay for identification of inhibitors of TNFa-specific NF-kB inductionconfirmatory
2503SAR analysis of Muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells - Set 2confirmatory
2505SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cells - Set 2confirmatory
2789SAR analysis of NF-kappaB dependent luciferase using Doxorucibin as an inducer - Set 2confirmatory
2792SAR analysis of NF-kappaB dependent luciferase using PMA/Ionomycin as an inducer - 2confirmatory
2793SAR analysis of NF-kappaB dependent luciferase using DAP as an inducer - Set 2confirmatory
2798SAR analysis of compounds that inhibit NOD1 - Set 3confirmatory
2799SAR analysis of compounds that inhibit NOD2 - Set 3confirmatory
2800SAR analysis of compounds that are cytotoxic to HEK293 - Set 3confirmatory
2801SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 3confirmatory
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 HTS assay that utilizes NF-kB-mediated luciferase reporter gene activity as a measure of NOD1 modulation. The assay uses a luminescent readout.

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)

Primary Screen and Single-concentration confirmation
Day 1 Procedure
1) Harvest HEK-293-T NFKB-Luc at 100% confluency
2) Dispense 3 uL (6000 cells)/well to every well of a 1536 TC-treated white plate (Corning # 3727).
3) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.
4) Using a HighRes biosolution pintool equipped with V&P Scientific pins, stamp 10nl of 2mM cmpds in DMSO (col 5-48) and 10nl DMSO controls (col 1-4) to plates
5) Lid Plates. Incubate cells for 1 hour at room temp.
6) Dispense 2 uL/well of g-tri-DAP (1.875 ug/mL) in assay media containing 1.375% DMSO to columns 3-48.
7) Spin down plates for 30 sec in an Eppendorf 5810 centrifuge.
8) Lid Plates. Incubate overnight (16 hours) in 37 oC 5% CO2 incubator

Day 2 Procedure
1) Equibrate plates to room temp for 10 min.
2) Add 3 uL SteadyGlo well with Multidrop
3) Spin plates for 10 sec in a Velocity11 VSpin, shake for 30 sec.
4) Incubate plates for 20 min at toom temp.
5) Read luminescence on Perkin Elmer Viewlux.

NOD1 Dose Response assay

(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 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 gamma-tri-DAP to cell suspension at 0.75 ug/mL.
6) Seed 13000 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 SteadyGlo 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 with a Z score of less than or equal to -3 at 4 uM concentration are defined as actives in the primary screen.

ZScore is calculated as (wellValue - Mean) / StdDev. Where the mean is the mean of all of the valid wells on the plate and the STDDev is the standard deviation of all of the wells on the plate

These compounds were retested in a single concentration confirmation screen in both NOD1 assay and an assay detecting non-specific NF-kB activation by TNF-alpha (AID 1852).

Compounds with an average of >= 50% inhibition in the NOD1 reconfirmation assay are considered active.

Compounds that were active in the NOD 1 assay while showing no activity in TNF-alpha assay were subsequently run in dose response mode. Compounds are considered active if the EC50 < 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. The score is correlated with the zScore in the assay demonstrated by a compound at 4 uM concentration:
a. If the compound is determined to be inactive, zScore > -3 then the assigned score is 0
b. If the compound is determined to be active, zScore <= -3 then the assigned score is 30.

Samples retested in the single concentration screen were scored as follows:

a. If the Ave %Efficacy of Repeats is less than 0%, then the assigned score is 0
b. If primary Ave %Efficacy of Repeats is greater than 100%, then the assigned score is 40
c. If primary Ave %Efficacy of Repeats is between 0% and 100%, then the calculated score is (Ave %Efficacy of Repeats)*0.4

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*(pEC50 - 3)*QC,
where pEC50 is a negative log(10) of the EC50 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
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_QualifierThis qualifier is to be used with the next TID, EC50. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
2EC50*EC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(EC50)Standard Error of EC50 valueFloat
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Ave %Efficacy of Repeats at 4 uM (4μM**)Average of the % Efficacy of the cherry picked primary hits retested in duplicate at 4 uMFloat%
6Std.Err(Repeats) (4μM**)Standard error of the % Efficacy of the cherry picked primary hits retested in duplicate at 4 uMFloat
7%Efficacy at 4 uM (4μM**)% Efficacy in primary screeningFloat%
8zScoreThe zScore in primary screeningFloat
9Mean HighMean Absorbance of negative controls in the corresponding plateFloatOD
10STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatOD
11Mean LowMean Absorbance of positive controls in the corresponding plateFloatOD
12STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatOD

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

Data Table (Concise)
Classification
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