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 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 cytotoxity assay that could be multiplexed with one or more of the following assays:
1) NOD 1, the protocol is described in AID 1578.
2) NOD2, the protocol is described in AID 2001.
3) TNFa, the protocol is described in AID 1852.

The purpose was to gauge cytotoxity in actives emerging from the NOD1 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.

Protocol:
Assay materials:
1) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory.
2) gamma-tri-DAP (Ana Spec cat #60774) obtained from assay provider's laboratory.
3) Resazurin (Sigma cat# R7017-5G)

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

This assay could be multiplexed with one or more of the following assays:
1) NOD1, AID 1578.
2) NOD2, AID 2001.
3) TNFa, AID 1852.

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 with Labcyte Echo 550 50 nl total volume 100% DMSO (DPI compounds columns 5-46 & DMSO controls col 1-4, 47-48) to 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 20 nL Resazurin 2.2 mM 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. (Bodipy dichroic, 595nM emission, 531nM excitation filter).

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