|uHTS luminescence assay for the identification of compounds that inhibit NOD2 in MDP treated cells - BioAssay Summary
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
BioActive Compounds: 263
Depositor Specified Assays
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.
In development of an earlier assay, AID 1566, it was found that MDP did not adequately stimulate NOD2 in Hek293-T KbLuc cell for testing with 4 uM compounds. MDP is useable in a dose response assay without the need for NOD2OE cells.
The assay described below is a cell-based HTS assay that utilizes NF-kB-mediated luciferase reporter gene activity as a measure of NOD2 modulation. The HEK-293-T NF-kB cell line designed for luminescent detection of NF-kB induction was utilized in this assay. The assay uses a luminescent readout.
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.
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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.
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1. Hek293-T KbLuc cell line obtained from the assay provider's laboratory.
2. BrightGlo (Promega)
3. MDP (muramyl dipeptide)
NOD2 Dose Response
(This assay was multiplexed with a cytotoxicity protocol described in AID 1848).
1. Harvest HEK-293-T NF-kB -Luc at 100% confluency
2. Add 1 uL/well NOD assay media with Multidrop
3. Spin down plates @ 1000 RPM for 1 min.
4. Add with Echo 50nl total volume 100% DMSO to plates.
*10pt serial dilution with Dilution Factor 2, starting from 20uM or 5uM Final assay concentrations. 1% DMSO Final assay concentration in 5uL.
5. Make cells suspension at 1.25X for final seeding density of 6000 cells/well in 4uL well volume.
6. Add MDP to 1.25X cell suspension at 0.875ug/mL.
7. Seed 6000 cells/well in 4uL/well to full plate HEK-293-T NF-kB to Corning # 3727 wht, 1536, hi-prof, TC-treated plate.
8. Spin down plates @ 500 RPM for 5 min.
9. Lid Plates. Sandwich each set 4 of four plates between two lidded 384 plates filled with H2O.
10. Wrap plates securely in single layer of Saran Wrap (PVDC).
11. Incubate overnight (14 hours) in 37 oC 5% CO2 incubator
1. Add 3 ul BrightGlo well with Multidrop
2. spin plates @ 1000 RPM for 1 min, shake 20 min
3. Read luminescence on Viewlux
Compounds with IC50 <=10uM and Hill slope 2.5 >= nH >= 0.5 are defined as active in this assay. Compounds that were active in the NOD 2 assay were subsequently run in TNF alpha dose response mode (AID 1852)
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 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
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
* Activity Concentration.
Data Table (Concise)