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-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.

This dose response assay is developed and performed to confirm hits originally identified in "uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assay" AID 1849 and "uHTS Fluorescence assay for the identification of cytotoxic compounds" among compounds active in NOD2 cell inhibition assay 1848 and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

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.

This assay (AID 2056) could be multiplexed with one or more of the following assays:
1) NOD1, the protocol is described in AID 2072.
2) NOD2, the protocol is described in AID 2054.
3) TNFa, the protocol is described in AID 1980.

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 was multiplexed with a NOD1 and NOD2 protocol described in AID 1578 and AID 1566).

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).

Compounds were tested in 1 or more ranges.

Range1 0-20 uM
Range2 0-5 uM

Compounds are considered active if the IC50_Mean < 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
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 to this assay

2) 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. 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.
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 this 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 = 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.

Grant Number: 1 R03 MH084844-01