Single concentration confirmation of uHTS hits for cystic fibrosis induced NFkb Inhibitors in a fluoresence assay
The inflammatory response in Cystic Fibrosis (CF) is a complex interplay between several factors. It has been suggested that pro-inflammatory cytokines are elevated in the epithelial lining fluid samples of CF patients(1). Conversely, expressions of anti-inflammatory cytokines are reduced(2). 90% of all CF deaths occurs from defective lung function. The activation of NFkB via toll-like receptors following bacterial infection is principally involved in the regulation of lung inflammation in CF(3,4) ..more
BioActive Compounds: 833
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: IR21 NS061743-01
Assay Provider: Rangan Maitra, Ph.D., RTI International University
The inflammatory response in Cystic Fibrosis (CF) is a complex interplay between several factors. It has been suggested that pro-inflammatory cytokines are elevated in the epithelial lining fluid samples of CF patients(1). Conversely, expressions of anti-inflammatory cytokines are reduced(2). 90% of all CF deaths occurs from defective lung function. The activation of NFkB via toll-like receptors following bacterial infection is principally involved in the regulation of lung inflammation in CF(3,4)
This project proposes to develop and optimize a Pseudomonas aeruginosa filtrate (PAF)-induced NFkB-GFP reporter assay using immortalized CF airway epithelial cell line (KKLEB) and screen the MLPCN library for inhibitors of PAF-induced NFkB pathway and distinguish compounds that are inhibiting via the toll-like receptor NFkB pathway to treat lung inflammation in CF.
This purpose of this assay is to confirm hits from "uHTS identification of cystic fibrosis induced NFkb Inhibitors in a fluoresence assay." AID 588850.
1. Bonfield, T.L., Konstan, M. W. & Berger, M. Altered respiratory epithelial cell cytokine production in cystic fibrosis. J Allergy Clin Immunol 104, 72-8 (1999).
2. Bonfield, T.L et al. Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. Am J Respir Cell Mol Biol 13, 257-61 (1995).
3. Zhang, Z., Louboutin, J.P., Weiner, D.J., Goldberg, J.B. & Wilson, J. M. Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by toll-like receptor 5. Infect Immunol 73, 7151-60 (2005).
4. Greene, C. M. et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J. Immunol 174, 1638-46 (2005).
KKLEB-NFkB-GFP cells (Assay Provider)
Pseudomonas aeruginosa filtrate (PAF) (Assay Provider)
Fetal Bovine Serum (Hyclone SH30396.03)
Penicillin Streptomycin solution
TrypLE (Invitrogen 12563)
DPBS without calcium and magnesium (1X)
Corning culture flasks
Black CellBind 1536-well plates (Corning 3833)
I. Cell Suspension
1- Dispense 3 uL/well of cells at 5X10;5 cells/mL to the whole plate (plate cells in 2% FBS assay media).
2- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.
II. Compound Addition:
3- Transfer test compounds to columns 5-48 and DMSO to columns 1-4 using the Labcyte ECHO 555.
4- Transfer volume of test compound and DMSO is 2.5nL of 10mM DPI reorder plate making 5uM compound concentration at 0.04% DMSO final.
5-Spin down plates on Vspin at 1000 rpm for 1 minute.
6-Put Kalypsys metal lids on plates, incubate plates at 37 degrees C with 5% CO2 for 2 hours.
III. Reagent Addition
7- Dispense 3 uL/well of serum free assay media to columns 1 and 2.
8- Dispense 3 uL/well of PAF (dilute in serum free assay media) to columns 3-48.
9- Spin down plates without lids on Vspin at 2000 rpm for 2 min
10- Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.
IV. Reading plates:
11-Spin plates upside down with a container at 1000 rpm for 15 sec. Dab them with a tissue to dry them and Read immediately on envision for GFP fluorescence.
Compounds that demonstrated a %Activity_mean of >= 50% at 5 uM concentration are defined as actives in this assay.
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 and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)