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BioAssay: AID 2798

SAR analysis of compounds that inhibit NOD1 - Set 3

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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Active(16)
 
 
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AID: 2798
Data Source: Burnham Center for Chemical Genomics (BCCG-A345-NOD1-DryPowder-Assay-3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-04-29
Hold-until Date: 2010-09-14
Modify Date: 2011-01-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 16
Depositor Specified Assays
AIDNameTypeProbeComment
1566uHTS luminescence assay for the identification of compounds that inhibit NOD2confirmatory
1575Summary assay for the identification of compounds that inhibit NOD1summary2
1578uHTS luminescence assay for the identification of compounds that inhibit NOD1confirmatory
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
2333SAR analysis of compounds that inhibit NOD1 revisedconfirmatory
2335SAR analysis of compounds that are cytotoxic to HEK293 revisedconfirmatory
2466SAR analysis of compounds that inhibit NOD1 - Set 2confirmatory
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.

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.

This assay could be multiplexed with a HEK-293-T cytotoxicity assay, AID 2335.

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 pending).

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 were tested in 1 or more ranges.

Range1 0-20 uM

Compounds are considered active if the IC50_Mean < 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 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. 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 NOD1 inhibition 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 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.
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_1_Range1This 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_1_Range1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1_Range1Standard Error of 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 20 uM_first_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
9% Activity at 10 uM_first_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
10% Activity at 5 uM_first_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
11% Activity at 2.5 uM_first_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
12% Activity at 1.25 uM_first_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
13% Activity at 0.625 uM_first_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
14% Activity at 0.3125 uM_first_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
15% Activity at 0.15625 uM_first_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
16% Activity at 0.078125 uM_first_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
17% Activity at 0.0390625 uM_first_point_Range1 (0.0390625μM**)% Inhibition at a given 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 20 uM_second_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
20% Activity at 10 uM_second_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
21% Activity at 5 uM_second_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
22% Activity at 2.5 uM_second_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
23% Activity at 1.25 uM_second_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
24% Activity at 0.625 uM_second_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
25% Activity at 0.3125 uM_second_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
26% Activity at 0.15625 uM_second_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
27% Activity at 0.078125 uM_second_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
28% Activity at 0.0390625 uM_second_point_Range1 (0.0390625μM**)% Inhibition at a given concentrationFloat%
29IC50_Qualifier_2_Range1This 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_2_Range1IC50 value determined using sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2_Range1Standard Error of IC50 valueFloatμM
32nH_2_Range1Hill coefficient determined using sigmoidal dose response equationFloatμM
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 20 uM_third_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
35% Activity at 10 uM_third_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
36% Activity at 5 uM_third_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
37% Activity at 2.5 uM_third_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
38% Activity at 1.25 uM_third_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
39% Activity at 0.625 uM_third_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
40% Activity at 0.3125 uM_third_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
41% Activity at 0.15625 uM_third_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
42% Activity at 0.078125 uM_third_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
43% Activity at 0.0390625 uM_third_point_Range1 (0.0390625μM**)% Inhibition at a given 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 20 uM_fourth_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
46% Activity at 10 uM_fourth_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
47% Activity at 5 uM_fourth_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
48% Activity at 2.5 uM_fourth_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
49% Activity at 1.25 uM_fourth_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
50% Activity at 0.625 uM_fourth_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
51% Activity at 0.3125 uM_fourth_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
52% Activity at 0.15625 uM_fourth_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
53% Activity at 0.078125 uM_fourth_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
54% Activity at 0.0390625 uM_fourth_point_Range1 (0.0390625μM**)% Inhibition at a given concentrationFloat%

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

Data Table (Concise)
Classification
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