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BioAssay: AID 624344

Dose Response confirmation of uHTS small molecule hits for cystic fibrosis induced NFkb Inhibitors in a panel 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
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 Tested Compounds
 Tested Compounds
All(737)
 
 
Active(172)
 
 
Inactive(565)
 
 
 Tested Substances
 Tested Substances
All(737)
 
 
Active(172)
 
 
Inactive(565)
 
 
AID: 624344
Data Source: Burnham Center for Chemical Genomics (SBCCG-A856-CF-PAF-Panel-Assay)
BioAssay Type: Panel, Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 172
Related Experiments
AIDNameTypeComment
588850uHTS identification of cystic fibrosis induced NFkb Inhibitors in a fluoresence assayScreeningdepositor-specified cross reference
588858Summary assay for small molecule cystic fibrosis induced NFkb InhibitorsSummarydepositor-specified cross reference
602141uHTS determination of small molecule cytotoxicity in a fluorescence assay to identify cystic fibrosis induced NFkb InhibitorsScreeningsame project related to Summary assay
602472Single concentration confirmation of uHTS hits for cystic fibrosis induced NFkb Inhibitors in a fluoresence assayScreeningsame project related to Summary assay
624343Dose Response confirmation of uHTS small molecule hits for cystic fibrosis induced NFkb Inhibitors in a PAF-induced IL8 counterscreenConfirmatorysame project related to Summary assay
624347Dose Response confirmation of uHTS small molecule hits for cystic fibrosis induced NFkb Inhibitors in a TNFa-induced IL8 counterscreenConfirmatorysame project related to Summary assay
651592SAR analysis of molecule cystic fibrosis induced NFkb Inhibitors in a panel assayConfirmatorysame project related to Summary assay
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: 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 assay is a follow-up to "Single concentration confirmation of uHTS hits for cystic fibrosis induced NFkb Inhibitors in a fluorescence assay multiplexed with luminescence cytotoxicity assay." AID 588850. Compounds active in the primary fluorescence assay and inactive in the luminescence cytotoxicity 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).
Panel Information
CF-PAF panel - panel
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1IC50_CF-PAF_Mean68750cystic fibrosis transmembrane conductance regulator [Homo sapiens] [gi:90421313]
Mean IC50 value
2IC50_1_CF-PAFcystic fibrosis transmembrane conductance regulator [Homo sapiens] [gi:90421313]
IC50 value determined using a sigmoidal dose response equation
3IC50_2_CF-PAFcystic fibrosis transmembrane conductance regulator [Homo sapiens] [gi:90421313]
IC50 value determined using a sigmoidal dose response equation
4IC50_Cytotoxicity_Mean534203Mean IC50 value
5IC50_1_CytotoxicityIC50 value determined using a sigmoidal dose response equation
6IC50_1_CytotoxicityIC50 value determined using a sigmoidal dose response equation

§ Panel component ID.
Protocol
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)

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 10mM 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 5X10e5 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- Dispense 3 uL/well of 13.33% LB (dilute in serum free assay media) to columns 1-4 final LB concentration is 5%.
6- Dispense 3 uL/well of 13.33% PAF (dilute in serum free assay media) to columns 5-48, final PAF concentration is 5%.
7- Spin down plates Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.
8- Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.

III. Reading plates:
9- 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.
10-Add 3ul ATPLite using combi dispenser.
11-Spin plates 2000 rpm for 2 min.
12-Read on ViewLux for luminescence.
Comment
Compounds that demonstrated an IC50 of <= 20 uM in the florescence assay and demonstrated an IC50 of > 50 uM in the luminescence cytotoxicty assay 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
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome_CF-PAFActivity outcome for the CF-PAF portion of the panel1cystic fibrosis transmembrane conductance regulator [Homo sapiens]Outcome
2PUBCHEM_ACTIVITY_SCORE_CF-PAFActivity Score for the CF-PAF portion of the panel1Integer
3IC50_CF-PAF_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value1String
4IC50_CF-PAF_Mean*Mean IC50 value for the CF-PAF replicates determined using a sigmoidal dose response equation1FloatμM
5Outcome_CytotoxicityActivity outcome for the cytotoxicity portion of the panel4Outcome
6PUBCHEM_ACTIVITY_SCORE_CYTOTOXICITYActivity Score for the cytotoxicity portion of the panel4Integer
7IC50_Cytotoxicity_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value4String
8IC50_Cytotoxicity_MeanMean IC50 value for the cytotoxicity replicates determined using a sigmoidal dose response equation4FloatμM
9IC50_Qualifier_1_CF-PAFThis qualifier is to be used with the next TID, . If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value2cystic fibrosis transmembrane conductance regulator [Homo sapiens]String
10IC50_1_CF-PAFIC50 value determined using a sigmoidal dose response equation2FloatμM
11Std.Err(IC50)_1_CF-PAFStandard Error of the IC50 value2FloatμM
12nH_1_CF-PAFHill coefficient determined using sigmoidal dose response equation2Float
13Excluded_Points_first_point_Range1_CF-PAFFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.2String
14% Activity at 79 uM_first_point_Range1_CF-PAF (79μM**)% Activity at the test concentration2Float%
15% Activity at 39.5 uM_first_point_Range1_CF-PAF (39.5μM**)% Activity at the test concentration2Float%
16% Activity at 19.75 uM_first_point_Range1_CF-PAF (19.75μM**)% Activity at the test concentration2Float%
17% Activity at 9.875 uM_first_point_Range1_CF-PAF (9.875μM**)% Activity at the test concentration2Float%
18% Activity at 4.9375 uM_first_point_Range1_CF-PAF (4.9375μM**)% Activity at the test concentration2Float%
19% Activity at 2.46875 uM_first_point_Range1_CF-PAF (2.46875μM**)% Activity at the test concentration2Float%
20% Activity at 1.234375 uM_first_point_Range1_CF-PAF (1.23438μM**)% Activity at the test concentration2Float%
21% Activity at 0.6171875 uM_first_point_Range1_CF-PAF (0.617188μM**)% Activity at the test concentration2Float%
22% Activity at 0.3085938 uM_first_point_Range1_CF-PAF (0.308594μM**)% Activity at the test concentration2Float%
23% Activity at 0.1542969 uM_first_point_Range1_CF-PAF (0.154297μM**)% Activity at the test concentration2Float%
24IC50_Qualifier_2_CF-PAFThis qualifier is to be used with the next TID, . If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value3cystic fibrosis transmembrane conductance regulator [Homo sapiens]String
25IC50_2_CF-PAFIC50 value determined using a sigmoidal dose response equation3FloatμM
26Std.Err(IC50)_2_CF-PAFStandard Error of the IC50 value3FloatμM
27nH_2_CF-PAFHill coefficient determined using sigmoidal dose response equation3Float
28Excluded_Points_second_point_Range1_CF-PAFFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.3String
29% Activity at 79 uM_second_point_Range1_CF-PAF (79μM**)% Activity at the test concentration3Float%
30% Activity at 39.5 uM_second_point_Range1_CF-PAF (39.5μM**)% Activity at the test concentration3Float%
31% Activity at 19.75 uM_second_point_Range1_CF-PAF (19.75μM**)% Activity at the test concentration3Float%
32% Activity at 9.875 uM_second_point_Range1_CF-PAF (9.875μM**)% Activity at the test concentration3Float%
33% Activity at 4.9375 uM_second_point_Range1_CF-PAF (4.9375μM**)% Activity at the test concentration3Float%
34% Activity at 2.46875 uM_second_point_Range1_CF-PAF (2.46875μM**)% Activity at the test concentration3Float%
35% Activity at 1.234375 uM_second_point_Range1_CF-PAF (1.23438μM**)% Activity at the test concentration3Float%
36% Activity at 0.6171875 uM_second_point_Range1_CF-PAF (0.617188μM**)% Activity at the test concentration3Float%
37% Activity at 0.3085938 uM_second_point_Range1_CF-PAF (0.308594μM**)% Activity at the test concentration3Float%
38% Activity at 0.1542969 uM_second_point_Range1_CF-PAF (0.154297μM**)% Activity at the test concentration3Float%
39IC50_Qualifier_1_CytotoxicityThis qualifier is to be used with the next TID, . If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value5String
40IC50_1_CytotoxicityIC50 value determined using a sigmoidal dose response equation5FloatμM
41Std.Err(IC50)_1_CytotoxicityStandard Error of the IC50 value5FloatμM
42nH_1_CytotoxicityHill coefficient determined using sigmoidal dose response equation5Float
43Excluded_Points_first_point_Range1_CytotoxicityFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.5String
44% Activity at 79 uM_first_point_Range1_Cytotoxicity (79μM**)% Activity at the test concentration5Float%
45% Activity at 39.5 uM_first_point_Range1_Cytotoxicity (39.5μM**)% Activity at the test concentration5Float%
46% Activity at 19.75 uM_first_point_Range1_Cytotoxicity (19.75μM**)% Activity at the test concentration5Float%
47% Activity at 9.875 uM_first_point_Range1_Cytotoxicity (9.875μM**)% Activity at the test concentration5Float%
48% Activity at 4.9375 uM_first_point_Range1_Cytotoxicity (4.9375μM**)% Activity at the test concentration5Float%
49% Activity at 2.46875 uM_first_point_Range1_Cytotoxicity (2.46875μM**)% Activity at the test concentration5Float%
50% Activity at 1.234375 uM_first_point_Range1_Cytotoxicity (1.23438μM**)% Activity at the test concentration5Float%
51% Activity at 0.6171875 uM_first_point_Range1_Cytotoxicity (0.617188μM**)% Activity at the test concentration5Float%
52% Activity at 0.3085938 uM_first_point_Range1_Cytotoxicity (0.308594μM**)% Activity at the test concentration5Float%
53% Activity at 0.1542969 uM_first_point_Range1_Cytotoxicity (0.154297μM**)% Activity at the test concentration5Float%
54IC50_Qualifier_2_CytotoxicityThis qualifier is to be used with the next TID, . If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value6String
55IC50_2_CytotoxicityIC50 value determined using a sigmoidal dose response equation6FloatμM
56Std.Err(IC50)_2_CytotoxicityStandard Error of the IC50 value6FloatμM
57nH_2_CytotoxicityHill coefficient determined using sigmoidal dose response equation6Float
58Excluded_Points_second_point_Range1_CytotoxicityFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.6String
59% Activity at 79 uM_second_point_Range1_Cytotoxicity (79μM**)% Activity at the test concentration6Float%
60% Activity at 39.5 uM_second_point_Range1_Cytotoxicity (39.5μM**)% Activity at the test concentration6Float%
61% Activity at 19.75 uM_second_point_Range1_Cytotoxicity (19.75μM**)% Activity at the test concentration6Float%
62% Activity at 9.875 uM_second_point_Range1_Cytotoxicity (9.875μM**)% Activity at the test concentration6Float%
63% Activity at 4.9375 uM_second_point_Range1_Cytotoxicity (4.9375μM**)% Activity at the test concentration6Float%
64% Activity at 2.46875 uM_second_point_Range1_Cytotoxicity (2.46875μM**)% Activity at the test concentration6Float%
65% Activity at 1.234375 uM_second_point_Range1_Cytotoxicity (1.23438μM**)% Activity at the test concentration6Float%
66% Activity at 0.6171875 uM_second_point_Range1_Cytotoxicity (0.617188μM**)% Activity at the test concentration6Float%
67% Activity at 0.3085938 uM_second_point_Range1_Cytotoxicity (0.308594μM**)% Activity at the test concentration6Float%
68% Activity at 0.1542969 uM_second_point_Range1_Cytotoxicity (0.154297μM**)% Activity at the test concentration6Float%

* Activity Concentration. ** Test Concentration. § Panel component ID.
Additional Information
Grant Number: IR21 NS061743-01

Classification
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