Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation
NF-kB, NF-kappaB, transcription factor, antigen receptor, Protein Kinase C-theta, PMA, ionomycin, luciferase, luminescence ..more
BioActive Compounds: 128
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: The Burnham Institute
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal number: 1 X01 MH077633-01
NF-kB, NF-kappaB, transcription factor, antigen receptor, Protein Kinase C-theta, PMA, ionomycin, luciferase, luminescence
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.
 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
This assay utilizes HEK-293 cells that have been stably transfected with the mammalian expression plasmid pUC13-4xNFkB-Luc, allowing the expression of the firefly luciferase under the control of four tandem HIV NFkB response elements. As previously described, stimulation with a PMA/Ionomycin mix results in NF-kB activation, thus increasing luciferase expression and luminescence levels measured with an appropriate substrate. Compounds that decrease light emission are potential inhibitors and may be able to block the NF-kB activation triggered by the PMA/Ionomycin stimulation, hence providing valuable tools to decipher this new pathway.
The primary HTS assay was conducted in 1536-well format. All compounds were tested once at a 3 micromolar final concentration.
A mathematical algorithm was used to determine nominally active compounds.
Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater %inhibition than the cutoff parameter was declared active.
HEK-293 stably transfected with the pUC13-4xNFkB-Luc were cultured in T-175 flasks (Corning part#431080) at 37 degrees Celsius, 5%CO2 and 95% relative humidity.
The growth media consisted of Dulbecco's Modified Eagle's Media (Invitrogen part# 11965-092) supplemented with 10% v/v heat inactivated fetal bovine serum (Hyclone part#SH30088), 1% v/v penicillin-streptomycin mix (Invitrogen part#15140-122) and 1ug/mL puromycin (InvivoGen part#ant-pr-1).
Prior to the assay, cells were suspended to a concentration of 625,000 cells per milliliter in phenol red free Dulbecco's Modified Eagle's Media (Invitrogen part# 21063-029) supplemented with 10% v/v heat inactivated fetal bovine serum (Hyclone part#SH30088), 1% v/v penicillin-streptomycin mix (Invitrogen part#15140-122) and 1ug/mL puromycin (InvivoGen part#ant-pr-1).
The assay began by dispensing 4 microliters of cell suspension to each well (i.e. 2,500 cells/well) of a white solid-bottom 1536-well plate (Kalypsys part# K1536 SWST)
Plates were then placed in the incubator overnight at 37 degrees Celsius, 5%CO2 and 95% relative humidity.
Twenty four hours after seeding, cells were treated with 15 nL/well of compounds (3 micromolar, final DMSO concentration of 0.30%). One hour later, cells were stimulated with 1 microliter per well of a PBS mix of PMA (100ng/mL, Calbiochem part#524400) and Ionomycin (50ng/mL, Calbiochem part#407950). Plates were subsequently returned to the incubator for 16 hours (37 degrees Celsius, 5%CO2 and 95% relative humidity).
After the incubation, plates were equilibrated to room temperature for 20 minutes. A luciferase assay was performed by adding 5 microliters per well of the SteadyLite HTS reagent (Perkin Elmer part#6016989).
After a 15 minutes incubation time, light emission was measured (30s per plate) with the ViewLux reader (Perkin Elmer).
The percent inhibition of each compound has been calculated as follow:
%inhibition = (1-(test_compound - Median_positive_control)/(Median_positive_control - Median_negative_control)*100
with positive control : 10 micromolar of the reference inhibitor #5653914 (ChemBridge)
and negative control : DMSO only
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to:
toxic compounds, compounds that inhibit luciferase activity, compounds that non-specifically inhibit transcriptional activity.
Data Table (Concise)