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, Burham Medical Research Institute, San Diego CA

The assay described below is a cytotoxity assay that was a multiplexed together with the luminescent detection of luciferase reporter gene. We utilized the assay's data for detection of cytotoxic compounds among the hits identified in the primary reporter gene assay. In this particular case, NOD2-overexpessing cell were utilized. The purpose was to gauge cytotoxity in actives emerging from the NOD2 dose response assay. Resazurin detects cell viability by converting from a nonfluorescent dye to the red fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth. Continued cell growth maintains a reduced environment while inhibition of growth maintains an oxidized environment. Reduction related to growth causes the REDOX indicator to change from the oxidized (nonfluorescent, purple color) form to the reduced (fluorescent, red color) form. The fluorescent signal is monitored using 531 nm excitation wavelength and 595 nm emission wavelength.

Assay materials:
1) NOD2OE HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory.
2) Resazurin (Sigma cat# R7017-5G)

Dose response cytotoxity assay for NOD2OE HEK-293-T NF-kB-Luc cells

(This assay was multiplexed with a NOD2 protocol described in AID 1566).

Day 1 Procedure

1) Harvest NOD2OE HEK-293-T NF-kB-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 with Labcyte Echo 550 50-nl total volume 100% DMSO (DPI cmpds col 5-46 & DMSO controls col 1-4, 47 -48) to plates from step 2.
5) Seed 13000 cells/well in 4 uL/well to full plate NOD2OE HEK-293-T NF-kb-Luc to Corning # 3727 white, 1536, hi-profile, TC-treated plate.
6) Spin down plates @ 500 RPM for 5 min on Eppendorf 5810 centrifuge.
7) Lid Plates. 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 20 nL Resazurin 2.2mM Resarurin in 50%/50% DMSO/dH20 using HighRes biosolution pintool equipped with V&P Scientific pins.
2) Wrap and incubate plates 1 hr in 37 oC 5% CO2 incubator
3) Top read Fluorescence intensity on Envision

Any compound that exhibited IC50 <20 uM was considered active.

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 thus was not utilized in this assay.
2) Second tier (41-80 range) is reserved for dose-response.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information:
Score=44+6*(pIC50 - 3)
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.
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