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
BioActive Compounds: 2997
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.
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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.
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 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
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)