SAR analysis of compounds that inhibit NOD2 revised
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: 45
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 and over expressed NOD2 as a measure of NOD2 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 NOD2" (AID 1566) 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 2056.
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.
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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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1. Hek293-T KbLuc cell line obtained from the assay provider's laboratory.
2. BrightGlo (Promega)
3. MDP (muramyl dipeptide)
NOD2 Dose Response
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 were tested in 1 or more ranges.
Range1 0-20 uM
Range2 0-5 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 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 NOD2 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)