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BioAssay: AID 435012

Summary assay for the identification of chemical inhibitors of T-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 ..
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AID: 435012
Data Source: Burnham Center for Chemical Genomics (BCCG-A381-NFkB-JukratPMA-Summary-Assay)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-06-22
Modify Date: 2011-07-27
Related Experiments
Show more
AIDNameTypeComment
435003uHTS luminescence assay for the identification of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-specified cross reference: Primary Screen
435020Single concentration confirmation of chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activationScreeningdepositor-specified cross reference
489026Dose response cytotoxicity of uHTS chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489033Dose Response selectivity of uHTS chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489035Dose response confirmation of uHTS chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
489041Dose response counterscreen of uHTS chemical inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
493251SAR analysis of small molecule cytotoxicity in HEK293 cells for activators of NF-kappaB using a luminescence assayConfirmatorydepositor-specified cross reference
504487SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504514SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504517SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assayConfirmatorydepositor-specified cross reference
504664SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a 697B cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
504665SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a Jurkat cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
504671SAR Analysis of small molecule inhibitors of T-cell specific antigen receptor-induced NF-kB activation in a HEK-293T cell line using a luminescence assay - Set 2Confirmatorydepositor-specified cross reference
Description:
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 [1]. 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 T cell lines. Therefore, in this rescreening paradigm, the primary screen will be accomplished using an NF-kB-luciferase reporter cloned into a T-cell line (Jurkat). 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.


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
Protocol
Please see pertinent AIDs: 435003, 493251, 504487, 504514, 504517, 504664, 504665, 504671
Comment
Probe molecules are defined as the positives of this assay and assigned a score of 100. Testing has not progressed to the point where a probe molecule has been identified.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: Jurkat
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML237
PubChem Substance ID (SID) for probe 1: 103061890
PubChem Compound ID (CID) for probe 1: 4174238
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 190
Target for probe 1: NF-kappaB activation in B-cells
Disease relevance for probe 1: Autoimmune disease
Anti-target for probe 1: NFkappaB activation in T-cells; TNFalpha-mediated NFkappaB activation; Cytotoxicity
Fold selectivity for probe 1: >379
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK126176/ (ID: 3013520)
Grant number for probe 1: MH077633-01
MLP Probe ML# for probe 2: ML236
PubChem Substance ID (SID) for probe 2: 103061876
PubChem Compound ID (CID) for probe 2: 665654
IC50/EC50 (nM) for probe 2: 34.9
Fold selectivity for probe 2: >1524
Grant number for probe 2: MH077633-01
NCBI Book chapter title for probe 1: B-cell Specific Inhibitors of NF-?B Activation
Additional Information
Grant Number: 1 X01 MH077633-01

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