SAR analysis of NF-kB dependent luciferase using PMA/Ionomycin as an inducer
The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis. ..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 Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA
The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis.
NF-kB pathway activated by antigen receptors is critical for acquired (as opposed to innate) immunity, contributing to T- and B-lymphocyte activation, proliferation, survival, and effector functions. Dysregulated NF-kB activation in lymphocytes can contribute to development of autoimmunity, chronic inflammation, and lymphoid malignancy. NF-kB activation pathway linked to antigen receptors involves a cascade of adapter and signal transducing proteins that minimally include a CARMA family protein, Bcl-10, MALT (Paracaspase), TRAF6, Ubc13, and Caspase-8. Formation of this complex is initiated by PKC-mediated phosphorylation of CARMA proteins. In T and B cells, this pathway is initiated by Protein Kinase C(PKC)-theta and PKC-beta, respectively, leading ultimately to IKK activation through a mechanism possibly involving lysine 63-linked polyubiquitination of IKK-gamma. Thus, the antigen receptor pathway for NF-kB activation is initiated and concluded by activation of protein kinases--namely, PKCs and IKKs, respectively.
The assay described below is a cell-based counter screen assay that utilizes NF-kB-mediated luciferase reporter gene activity as a measure of NF-kB induction by PKC activation, using phorbol ester (phorbol myristic acetate [PMA]) and Ca.sup.2+-ionophore Ionomycin to achieve PKC activation. The assay uses a luminescent readout.
This dose response assay is developed and performed to confirm hits originally identified in "uHTS luminescence assay for the identification of compounds that inhibit NOD1" (AID 1578) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
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1) HEK-293-T NFKB-Luc cell line obtained from the assay provider's laboratory.
2) PMA (Sigma).
3) Ionomycin (Sigma)
4) SteadyGlo (Promega)
1. Compounds were diluted in DMEM 10% FBS plus Pen/Strep (culture medium), and 10 uL were added into respective wells (96-well culture plate) to reach desired final concentrations.
2. PMA/Ionomycin mixture was added, in a final concentration of 11 ng/ml, to a culture medium suspension of HEK-293-T NFKB-Luc cells (1,1x10+6 cells per mililiter).
3. Pre-induced HEK-293-T NFKB-Luc cells were added into the respective wells (90 ul per well) and kept at 37oC (10% CO2 incubator) for 18 hours.
4. After incubation, plated cells were equilibrated to room temperature for 15 minutes.
5.Luciferase assay was then performed by adding 40 microliters per well of the Steady-GloTM reagent. After a ten minute incubation at room temperature, light emission was measured with the AnalystTM Microplate reader (LJL Biosystems).
Compounds are considered active if IC50 < 25 uM.
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 to this assay
2) 2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to 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's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3),
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.
Data Table (Concise)