Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation
Name: Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation ..more
BioActive Compounds: 78
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)
Proposal number 1X01-MH077633-01
External Assay ID: NFkB_INH_LUMI_1536_IC50
Name: Dose-response cell-based assay for chemical inhibitors of antigen receptor-induced NF-kappaB 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 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 new 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
NF-kB, NF-kappaB, transcription factor, antigen receptor, Protein Kinase C-theta, PMA, ionomycin, luciferase, luminescence, Scripps
Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical inhibitors of antigen receptor-induced NF-kappaB activation" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID=465.
Among the 128 compounds identified during the primary screening, 112 were assessed in dose-response experiments in 10 point, 1:3 serial dilution starting at a nominal test concentration of 30 micromolar.
As with the primary screen, the 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 NF-kB response elements. Stimulation with a PMA/ionomycin mix results in NF-kB activation, thus increasing luciferase expression and luminescence levels as 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 dose-response assay was conducted in 1536-well plate format.
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.
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 test compounds or positive and negative controls (10 micromolar of Chembridge compound part#5653914 and DMSO, respectively). Each compound dilution was assayed in triplicate, for a nominal total of 30 data points per dose response.
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 then 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 with the ViewLux reader (Perkin Elmer).
The percent inhibition of each well has been calculated as follow:
%inhibition = 100 * (1-(median_positive_control - test_compound)/(median_positive_control - median_negative_control))
with positive control : reference inhibitor #5653914 (ChemBridge, 10 micromolar)and negative control : DMSO only
For each compound, percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept.
In cases where the highest concentration tested (i.e. 30 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 30 micromolar. Compounds with IC50 of greater than 10 micromolar were considered inactive; compounds with IC50 of equal to or less than 10 micromolar were considered active.
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 or activate transcriptional activity.
Categorized Comment - additional comments and annotations
Data Table (Concise)