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

Assay materials:
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: single concentration counterscreen

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) 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
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

Compounds with %Activity of >50% are defined as actives in this assay.

The scores were calculated using the Tiered Activity Scoring System was developed and implemented by SBCCG. Its utilization for the assay is described below.

Activity Scoring
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 %Activity in the assay demonstrated by a compound at 4 uM concentration:
a. If primary %Activity is less than 0%, then the assigned score is 0
b. If primary %Activity is greater than 100%, then the assigned score is 40
c. If primary %Activity is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data and thus does not apply here.
3) Third tier (81-100 range) is reserved for dry powder SAR compounds and thus does not apply here.

Grant Number: 1 R03 MH084844-01