SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 3
Multiple cellular stimuli acting through various pathways lead to NF-kB induction. The assay described below uses tumor necrosis factor alpha (TNFa), a canonical NF-kB inducer, and is designed for identification of hits specific to TNFa-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the primary screening of the library in NOD1- and NOD2-specific assays (AIDs 1578 and 1566). The HEK-293-T NF-kB-Luc cell line designed for luminescent detection of NF-kB induction was utilized in this assay. ..more
BioActive Compounds: 8
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
Multiple cellular stimuli acting through various pathways lead to NF-kB induction. The assay described below uses tumor necrosis factor alpha (TNFa), a canonical NF-kB inducer, and is designed for identification of hits specific to TNFa-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the primary screening of the library in NOD1- and NOD2-specific assays (AIDs 1578 and 1566). The HEK-293-T NF-kB-Luc cell line designed for luminescent detection of NF-kB induction was utilized in this assay.
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 "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 the HEK-293-T cytotoxicity assay, AID 2335.
1) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory.
2) RNDsystems (Cat # 210-TA-010/CF)
3) SteadyGlo (Promega)
TNF-a NF-kB induction assay: Dose Response
Day 1 Procedure
1) Harvest HEK-293-T NF-kB-Luc at 100% confluency at 100% confluency
2) Supplement NOD plating media with DMSO to a final concentration of 0.62% DMSO
3) Dispense 3 uL (6000 cells)/well to every well of a 1536 TC-treated white plate (Corning # 3727).
4) Spin down plates at 500 rpm for 5 min in an Eppendorf 5810 centrifuge.
5) Serial compound dilutions: dispense with Labcyte Echo 550 50nl total volume 100% DMSO
6) Add 2 ul/well 0.25ng/mL dH20 TNF-a to wells in columns 3-48 using Multidrop.
7) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O
8) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).
9) Incubate overnight (14 hours) in 37 oC 5% CO2 incubator
Day 2 Procedure
1) Add 3 ul/well SteadyGlo to each well with Multidrop.
2) Spin plates @ 1000 rpm for 1 minute on Eppendorf 5810
3) Read luminescence on Perkin-Elmer Viewlux
The compounds were tested in 1 or more concentration ranges.
Range1 0-20 uM
Range2 0-5 uM
For all samples in each range that resolved to an IC50 the results were averaged and reported as IC50_Range. The IC50 results for all of the IC50s were averaged to produce IC50_Mean.
Compounds with an IC50_Mean <= 20 uM are considered to be active in this assay.
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.
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 is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable to this assay.
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. Compounds with an IC50 <= 0.0391 are assigned a score of 92.
c. For the remaining compounds 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.
d. 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 TNF-a inhibition assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
e. 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)