uHTS luminescence assay for the identification of chemical inhibitors of B-cell specific antigen receptor-induced NF-kB activation
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 ..
BioActive Compounds: 1077
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 B-cell specific manner.
 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
Primary Screen and Single-concentration confirmation
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) Dispense 10 nl 100% DMSO compounds (col 5-48) & 10nL 100% DMSO controls (col 1-4) to plates with Echo 555 (Labcyte) acoustic dispenser
6) Incubate plate for 1 hour in 37 oC 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 37 oC 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 that demonstrated an inhibition of >= 35% at 4 uM concentration 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. The score is correlated with % inhibition in the assay demonstrated by a compound at 4 uM concentration:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
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 and is not applicable in this assay
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)