HTS dose response assay for identification of inhibitors of TNFa-specific NF-kB induction
Multiple cellular stimuli acting through various pathways lead to NF-kB induction. The assay described below uses tumor necrosis factor alpha (TNF-a), a canonical NF-kB inducer, and is designed for identification of hits specific to TNF-a-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 more ..
BioActive Compounds: 437
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries 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 (TNF-a), a canonical NF-kB inducer, and is designed for identification of hits specific to TNF-a-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 assay is the dose response follow-up to "HTS assay for identification of inhibitors of TNF-a-specific NF-kB induction", AID 1852.
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)
(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
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 TNFa to cell suspension at 0.1 ng/mL.
6) Seed 6000 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 BriteGlo 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 are considered active if the IC50 < 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 in this assay.
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*(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
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)