AID	Name	Type	Probe	Comment
465	Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation	screening		Primary Screen
435022	uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation	screening		Primary Screen
435025	Summary assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation	summary	2	Summary AID.
489022	Dose response counterscreen of uHTS chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assay	confirmatory		Counter Screen
504518	SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assay	confirmatory
504670	SAR Analysis of small molecule inhibitors of B-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assay - Set 2	confirmatory

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 X01 MH077633-01
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, allows for the identification of compounds that appear to inhibit NF-kB induction, but in actuality are merely cytotoxic. Therefore, we utilized this assay to assess the cytotoxicity of hits emerging from the NF-B primary screening of the library in B-Cell-specific assay (AID 435022).

This dose response assay was developed and is performed to counterscreen hits originally identified in "uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation" (AID 435022).

This assay can be multiplexed with the TNF-alpha NF-kB induction assay (AID 489022).

References

[1] Thome M. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat Rev Immunol. 2004 May;4(5):348-59. Review

[2] Ruland J, Duncan GS, Elia A, del Barco Barrantes I, Nguyen L, Plyte S, Millar DG, Bouchard D, Wakeham A, Ohashi PS, Mak TW. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure. Cell. 2001 Jan 12;104(1):33-42

[3] McAllister-Lucas LM, Inohara N, Lucas PC, Ruland J, Benito A, Li Q, Chen S, Chen FF, Yamaoka S, Verma IM, Mak TW, Nunez G. Bimp1, a MAGUK family member linking protein kinase C activation to Bcl10-mediated NF-kappaB induction. J Biol Chem. 2001 Aug 17;276(33):30589-97

[4] Ruefli-Brasse AA, French DM, Dixit VM. Regulation of NF-kappaB-dependent lymphocyte activation and development by paracaspase. Science. 2003 Nov 28;302(5650):1581-4

[5] Zhou H, Wertz I, O'Rourke K, Ultsch M, Seshagiri S, Eby M, Xiao W, Dixit VM. Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO. Nature. 2004 Jan 8;427(6970):167-71

Assay materials:
1) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory
2) Assay Media: DMEM with no phenol red (Gibco 31053 or Hyclone SH30585.02), 2mM L-glutamine (omega sci GS-60 200mM), 1mM Na-pyruvate (Sigma S8636-100ML 100mM), 50IU/ml Penn/Strep(omega scientific PS-20 5000 IU/mL)
3) Resazurin (Sigma-Aldrich R7017)
4) Assay plate: 1536-well, TC-treated, black plate (Corning 3893)

Cytotoxicity assay: Dose Response
Day 1 Procedure
1) Harvest HEK-293-T NF-kB-Luc at 100% confluency
2) Make cells suspension at 1X for final seeding density of 6000 cells/well in 5uL well volume.
3) Dispense 6000 cells/well in 5 uL of assay media into col. 5-48 of assay plate
4) Dispense 5 uL/well of assay media into col. 1-4 of assay plate as positive control.
5) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge
6) With LabCyte Echo, dispense varying volumes of test compounds in DMSO into assay plate wells (Col. 5-44) to achieve appropriate dose response concentrations and range. Backfill test compound wells with DMSO to equilibrate DMSO concentrations. Transfer equal volumes of DMSO to control wells (Col. 1-4, 44-48). Maximum concentration of DMSO should be 1%.
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) Dispense 20nL of 2mM Resazarin in DMSO (FAC is 8uM) with LabCyte Echo to all wells of assay plate
2) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O
3) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).
4) Incubate for 1 hour in 37 oC 5% CO2 incubator
5) Top read Fluorescence Intensity (Ex. 531, Em. 595nm) on Perkin-Elmer EnVision

Compounds with and IC50_Mean <= 20 uM are defined as actives in this assay.

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 in this assay.

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's 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- or pathway-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 all 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

Grant Number: 1 X01 MH077633-01