SAR analysis of tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells.
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 Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute
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.
The NOD proteins participate in the signaling events triggered by host recognition of specific motifs (mostly, muropeptides) in bacterial peptidoglycan (PG). Upon activation, NODs induce activation of NF-kB, a central regulator of immune response, inflammation, and apoptosis. NOD2 is a general bacterial sensor that participates in the innate immunity against Gram-positive bacteria (S. pneumoniae, L. monocytogenes), Gram-negative bacteria (S. typhimurium) and mycobacteria (M. tuberculosis), while NOD1 recognizes mainly Gram-negative bacteria (E. coli, Chlamydia, H. pylori). Prior studies have shown the muramyldipeptide (MDP), a PG component, stimulates NOD2 activation in cells, while Ala-gamma Glu-diaminopimelic acid (GM-tri-DAP) stimulates NOD1, thus providing convenient, synthetic ligands for activating the proteins in intact cells.
Interleukin-8 (IL-8) is an important mediator of the immune reaction and a major chemokine involved in inflammatory responses. Apart of being a prototypical NF-kappaB target gene, recent studies have indicated that Gamma-tri-DAP induction of human breast cancer epithelial cell lines MCF-7 over-expressing NOD1, combined with small doses of cycloheximide (CHX), specifically induces IL-8 secretion (da Silva Correia, 2006; da Silva Correia 2007).
The assay described below uses tumor necrosis factor alpha (TNF-alpha), a canonical NF-kB inducer, and is designed for identification of hits specific to TNFa-modulated pathways. We utilized this assay to assess selectivity of hits emerging from the NOD1- specific assay, "SAR analysis of GM-Tri-DAP induced IL-8 production in MCF-7/NOD1 cells" (AID 2250) and NOD2- specific assay, "SAR analysis of Muramyl dipeptide (MDP) induced IL-8 production in MCF-7/NOD2 cells"(AID 2260) using IL-8.
Girardin SE, Jehanno M, Mengin-Lecreulx D, Sansonetti PJ, Alzari PM, Philpott DJ.Identification of the critical residues involved in peptidoglycan detection by Nod1. J Biol Chem. 2005 Nov 18;280(46):38648-56.
da Silva Correia, J., Miranda, Y., Leonard, N., Hsu, J., and Ulevitch, R. J. Regulation of Nod1-mediated signaling pathways. Cell Death Differ, 14: 830-839, 2007.
da Silva Correia, J., Miranda, Y., Austin-Brown, N., Hsu, J., Mathison, J., Xiang, R., Zhou, H., Li, Q., Han, J., and Ulevitch, R. J. Nod1-dependent control of tumor growth. Proc Natl Acad Sci U S A, 103: 1840-1845, 2006
1. MCF7 cells stably overexpressing NOD1
2. TNF-alpha (Sigma)
3. IL-8 OptEIA ELISA set (BD Biosciences, San Diego, CA)
4. SpectraMax 190 spectrophotometer (Molecular Devices)
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. TNF-alpha was added, in a final concentration of 5.4 ng/ml, to a culture medium suspension of MCF-7 cells (3,6x10+5 cells per mililiter).
3. Pre-induced MCF-7 cells were added into the respective wells (140 ul per well) and kept at 37oC (5% CO2 incubator) for 24 hours.
4. Thirty microliters of culture medium was used to measure IL-8 production using an IL-8 ELISA kit from BD according to the manufacturer's protocol. Briefly, 96-well ELISA plates (BD Biosciences) were coated with 100 uL per well of Capture Antibody diluted in Coating Buffer (100 mM Sodium carbonate, pH9.5) for overnight at 4oC. The wells were washed 3 times with 200 uL Washing Buffer (PBS with 0.05% Tween-20) and then blocked by 200 uL Assay Diluent (PBS with 10 % FBS) at room temperature for 1 h. The wells were then washed 3 times and 30 uL sample and proper amount of standards were added. The plate was sealed and incubated at 4oC overnight. After 5 total washes, 100 uL of Working Detector (detection antibody + SAv-HRP reagent) was added. The plate was then sealed and incubated for 1 h. After 7 total washes, 100 uL Substrate Solution was added and the plate was incubated for 30 min at room temperature in the dark. 50 uL Stop Solution was added and absorbance was read at 450 nm within 30 minutes on a spectrometer SpectraMax 190 (Molecular Devices). Absorbance at 570nm was subtracted from those of 450nm and data was analyzed.
5. Absorbance values were converted to IL-8 amounts (measured as pg/ml) according to standard curve analysis.
Compounds were tested in 1 or more ranges.
Range1 0-5 uM
Range2 0-25 uM
Compounds are considered active if they resolve to an IC50 and IC50_Mean < 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 50 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
Assay Cell Type: MCF-7
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration.
Data Table (Concise)