AID	Name	Type	Comment
588850	uHTS identification of cystic fibrosis induced NFkb Inhibitors in a fluoresence assay	screening
588858	Summary assay for small molecule cystic fibrosis induced NFkb Inhibitors	summary

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.

The purpose of this assay is to confirm hits from "uHTS identification of cystic fibrosis induced NFkb Inhibitors in a fluoresence assay." AID 588850. This PAF-induced IL8 secretion assay is a confirmatory assay to the primary assay to detect functional response downstream of PAF-induced NFKb. Compounds active in this assay are desired.

REFERENCES
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).

Assay Materials:
KKLEB-NFkB-GFP cells (Assay Provider)
Pseudomonas aeruginosa filtrate (PAF) (Assay Provider)
F12 (Invitrogen 11765)
Fetal Bovine Serum (Hyclone SH30396.03)
Penicillin Streptomycin solution (Invitrogen 15140122)
L-glutamine (100X) (Invitrogen 25030081)
TrypLE (Invitrogen 12563)
DPBS without calcium and magnesium (1X) (Hyclone SH30028.02)
Corning tissue culture flasks
Black CellBind 1536-well plates (Corning 3833)
Human IL8 Kit (20,000 tests) (Cisbio 62IL8PEC)

I. Compound Addition and Cell Plating:
1- Transfer test compounds to columns 5-44 and DMSO to columns 1-4 and 45-48 using the Labcyte ECHO 555. Transfer volume of test compound is 40 nL, 20 nL, 10 nL, 5 nL, and 2.5 nL from 10 mM stock and 40 nL, 20 nL, 10 nL, 5 nL, and 2.5 nL from 0.3125 mM stock for dose response which DMSO backfill to total volume of 40 nL per well. Compound concentration range from 79 uM to 0.15 uM (1:2 dilution, 10 concentration). DMSO transfer volume is 40 nL, final DMSO concentration is 1%.
2- Dispense 3 uL/well of cells at 8.3X10e4 cells/mL to the whole plate (plate cells in 2% FBS assay media).
3- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.
4- Put Kalypsys metal lids on plates, incubate plates at 37 degrees C with 5% CO2 for 2 hours.

II. Reagent Addition:
5- Prepare antibodies according to Cisbio protocol and add LB or PAF making it 1.33% working solution.
6- Dispense 2 uL/well of antibodies mix with 1.33% LB to columns 1-4, final LB concentration is 0.5%.
7- Dispense 2 uL/well of antibodies mix with 1.33% PAF to columns 5-48, final PAF concentration is 0.5%.
8- Spin down plates Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.
9- Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.

III. Reading plates:
10- Add 5ul reconstitution buffer to all wells.
11- Spin down plates Eppendorf centrifuge 5810 at 2000 rpm for 2 minutes.
12- Read immediately on envision for FRET.

Compounds that demonstrated an IC50 of <= 10uM 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
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 and is not applicable in this assay

2) Second tier (41-80 range) is reserved for dose-response confirmation data

a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]

This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
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.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

Grant Number: IR21 NS061743-01