SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assay
Many cellular pathways leading to activation of NF-kB-family transcription factors have been identified to be participating in host-defense, immunity, inflammation, and cancer. Recently, a unique pathway activated by antigen receptors on T- and B-Lymphocytes has been revealed, involving a cascade of participating proteins that includes CARMA1 (BIMP3), Bcl-10, paracaspase (MALT1), TRAF6, and more ..
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
Many cellular pathways leading to activation of NF-kB-family transcription factors have been identified to be participating in host-defense, immunity, inflammation, and cancer. Recently, a unique pathway activated by antigen receptors on T- and B-Lymphocytes has been revealed, involving a cascade of participating proteins that includes CARMA1 (BIMP3), Bcl-10, paracaspase (MALT1), TRAF6, and Ubc13. This pathway is initiated by Protein Kinase C-theta, which induces phosphorylation of components of this signaling pathway . Based on experiments using siRNA and dominant-negative mutants, it has been determined that treatment of cells with the combination of phorbol ester PMA and the calcium-ionophore Ionomycin triggers this pathway, resulting in NF-kB activation [2-5]. Compounds able to block this stimulation will be useful research tools for analysis of the physiological roles of this NF-kB activation pathway.
This assay was originally assigned to the Scripps Research Institute Molecular Screening Center (TSRI MSC) by the NIH during the MLSCN pilot phase ("Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation", AID 465.) In the original MLSCN screen a stably transfected reporter epithelial cell line (HEK293) that contains a luciferase gene driven by a NF-kB responsive promoter was used for compound library screening. The hits identified from that screen were very much less potent when tested in B cell lines. Therefore, in this rescreening paradigm, the primary screen will be accomplished using an NF-kB-luciferase reporter cloned into a human derived B-cell line (697). The goal therefore is to identify compounds that selectively inhibit one of the several known pathways that lead to NF-kB activation in mammalian cells in a T-cell specific manner.
The purpose of this assay is to confirm hits in "uHTS luminescence assay for the identification of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation"(AID 435003) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
 Thome M. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat Rev Immunol. 2004 May;4(5):348-59. Review
 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
 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
 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
 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
1) 697 NF-kB-Luc cell line obtained from Assay Provider
2) RPMI phenol red (-), Mediatech Car # 17105CV
3) Fetal Bovine Serum, Hyclone cat # SH30396.03HI
4) L-glutamine. Omega Scientific cat # GS-60
5) Na-pyruvate, Sigma cat # S8636-100M
6) Penn/Strep, Omega Scientific cat # PS-20
7) PMA, Calbiochem cat # 524400
8) Ionomycin, Calbiochem cat # 407950
9) SteadyGlo, Promega cat # E2550
Day 1 Procedure
1) Harvest 697 NF-kB-Luc at 2,000,000 cells/mL density. Spin, re-suspend in assay media, count.
2) Seed 15000 cells/well in 3 uL/well 697 NF-kB-Luc to full plate- Aurora 1536 low base white plate # 00029846 plate or non-TC-treated equivalent.
3) Plate with Kalypsys dispenser
4) Spin down plates @ 500 RPM for 1 min.
5) With a LabCyte Echo, dispense varying volumes of 10 mM test compounds in DMSO to achieve dose response concentrations and range (col 5-48) and backfill to equilibrate DMSO concentrations. To control wells (col 1-4), transfer an equal volume of DMSO.
6) Incubate plate for 1 hour in 37oC 5% CO2.
7) Add 2uL PMA/Ionomycin working dilution (6.5 ng/mL PMA/3.25 ng/mL Ionomycin) in assay media to col 3-48 and no agonist media to col 1 and 2 with Kalypsys dispenser.
8) Spin down plates @ 1000 RPM for 1 min.
9) Lid plates with Kalypsys plate lids.
10) Incubate plate overnight (16 hours) in 37oC 5% CO2 incubator.
Day 2 Procedure
1) Remove lid and incubate plate for 10 min in at room temp.
2) Add 3 ul/well SteadyGlo with Kalypsys dispenser
3) Spin plates @ 2000 RPM for 2 min, lid plate and incubate for 10 min at room temp.
4) Read luminescence on Envision Ultra sensitive Luminescent protocol for white Aurora 1536 low base white plate # 00029846
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 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 and is not applicable in 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 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 85 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)