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BioAssay: AID 445

IkB Signaling

Nuclear factor kappa-B (NF-kappa-B) plays an important role in normal B cell development and survival. Diffuse large B cell lymphoma (DLBCL) is the most commonly observed type of non-Hodgkin's lymphoma. Gene expression analysis has identified an activated B cell-like subtype of DLBCL (ABC-DLBCL) which expresses known NF-kappa-B target genes. In ABC-DLBCL cell lines this is due to high more ..
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 Tested Compounds
 Tested Compounds
All(111015)
 
 
Active(40)
 
 
Inactive(86445)
 
 
Inconclusive(24652)
 
 
 Tested Substances
 Tested Substances
All(112066)
 
 
Active(40)
 
 
Inactive(87312)
 
 
Inconclusive(24714)
 
 
 Related BioAssays
 Related BioAssays
AID: 445
Data Source: NCGC (ikb-signaling)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-07-14
Modify Date: 2007-04-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 40
Depositor Specified Assays
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AIDNameTypeComment
819Primary HTS assay for chemical potentiators of IL-1B stimulated NFkB nuclear translocationscreening
808Primary HTS assay for chemical potentiators of TNFalpha stimulated VCAM1 expressionscreening
1013Confirmatory Screen for chemical inhibitors of TNF alpha stimulated VCAM1 expressionconfirmatory
802Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expressionscreening
836Primary HTS assay for chemical modifiers of cytoskeleton assemblyscreening
1288Confirmatory Screen: Chemical Inhibitors of TNF alpha stimulated E Selectin expressionconfirmatory
1034Confirmatory Screen for chemical potentiatiors of TNF alpha stimulated VCAM1 expressionother
1249Confirmatory Screen for chemical modifiers of cytoskeleton assemblyconfirmatory
1246Primary HTS assay for chemical inhibitors of TNF alpha stimulated E-Selectin expressionscreening
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

NCGC Assay Overview:
Nuclear factor kappa-B (NF-kappa-B) plays an important role in normal B cell development and survival. Diffuse large B cell lymphoma (DLBCL) is the most commonly observed type of non-Hodgkin's lymphoma. Gene expression analysis has identified an activated B cell-like subtype of DLBCL (ABC-DLBCL) which expresses known NF-kappa-B target genes. In ABC-DLBCL cell lines this is due to high constitutive activity of I-kappa-B kinase (IKK), a key regulator of NF-kappa-B. Low molecular weight molecules that inhibit IKK have been shown to be selectively toxic for ABC-DLBCL cell lines. However, targets upstream of IKK have been largely unexplored and so nodes upstream of IKK in the NF-kappa-B pathway remain untested due to the lack of chemical probes.

A cell-sensor assay for I-kappa-B-alpha stabilization was developed with NIH investigator Dr. R. Eric Davis in Dr. Louis M. Staudt's laboratory. The assay uses green (CBG68) and red (CBR) emitting beetle luciferases [Chroma-Glo(TM) developed by Promega Corp.] where the green luciferase is fused to I-kappa-B-alpha and the red luciferase is present in its native state. Both luciferase genes are stably present in the OCI-Ly3 human ABC DLBCL cell line, but their expression is under the control of tetracycline-inducible promoters. Following the addition of doxycycline to induce expression of the luciferases, the green luminescence in control cultures rises only slowly because the CBG fusion partner I-kappa-B-alpha is targeted by IKK activity for rapid degradation. In contrast, green luminescence rises rapidly in the presence of proteasome inhibitors or IKK inhibitors. The rise in red luminescence of CBR is not affected by proteasome or IKK inhibitors, and serves as normalization for cell number and nonspecific effects. Compounds were screened as a concentration-titration series that ranged from 57 uM to 0.7 nM.
Protocol
NCGC Assay Protocol Summary:

Cells were seeded in 1536-well plates at 5000 cells/3ul in IMDM medium containing 0.5% FBS, w/o phenol red, L-glutamine, 25 mM HEPES, 3 mg/ml sodium bicarbonate. After centrifugation for 1 min. at 1000 rpm, 23 nl of compounds or DMSO were delivered to each well using a pin tool. Then 1ul 80ng/ml doxycycline was dispensed into white solid 1536-well plates and the plates were incubated at 37C/5% C02 in a cell incubator for 4 hrs. Then 4 ul Chroma-Glo luminescent substrate mix (Promega) was added to each well. The plate was incubated at R.T. for 10-15 min. The plates were measured on a ViewLux plate reader for green luminescence (540/20 nm filter) and red luminescence (618/8 nm filter). The green and red luminescent signals were corrected for red and green luminescence filter overlap using previously determined filter calibration constants (determined using the procedure outlined in the Promega Technical Manual). The %Activity was determined from the ratio of corrected green/red luminescent values. Green luminescent and ratio %Activity was determined by normalizing to the difference in signal between basal cells (0% Activity) and cells incubated with 10 uM of the proteasome inhibitor MG-132 (100% Activity). Red luminescent %Activity was normalized to the difference between red luminesence in basal cells (0%Activity) and zero luminescence (-100% Activity).

Concentration-response curves were fitted to the signals arising from the green luminescence (I-kappa-B-alpha-luciferase fusion; IkB-luc) and the red luminescence (unfused luciferase; luc) as well as the calculated ratio. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. Compounds were then categorized based on their concentration-response curves for all three datasets (Ikb-luc, luc and ratio). Active compounds showed concentration-dependent increases in the Ikb-luc and ratio data with little or no effect on the luc data. Signal Activators showed concentration-dependent increases in both the Ikb-luc and luc data with either no effect or a modest effect on the ratio data. Signal Inhibitors showed concentration-dependent decreases in either the Ikb-luc or luc datasets with either no effect or a concentration-dependent increase in the ratio data. Inactive compounds showed no effect in all three datasets.

Keywords: IKK, I-kappa-B, IkB, IkBa, cytotoxicity, luminescence, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
Comment
Compound Ranking:

1. Compounds are first classified as being probable IkB actives (99-50), signal activators (10), signal inhibitors (10) or inactive (0).

2. Within the IkB actives, compounds were ranked by efficacy and potency.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: IkB active, signal activator, signal inhibitor, inactive. IkB active compounds selectively enhanced luminesence of the IkB-luc channel. Signal activators and inhibitors nonselectively affected luminesence of both IkB-luc and luc channels.String
2PotencyConcentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.String
3Efficacy (% of Control)Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Phenotype ClassA description of how strongly a compound is associated with its phenotype. The integer denotes the phenotype, the mantissa denotes how strongly it is associated with that phenotype based on the IkB-luc, luc, and ratio dose-response curves. A smaller mantissa is more strongly associated.String
7ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
8ratio-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
9ratio-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
10ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
11ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
12ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13ratio-Excluded PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
14ratio-Activity at 0.70 nM% Activity of ratio channel at given concentration.Float%
15ratio-Activity at 1.6 nM% Activity of ratio channel at given concentration.Float%
16ratio-Activity at 3.7 nM% Activity of ratio channel at given concentration.Float%
17ratio-Activity at 8.2 nM% Activity of ratio channel at given concentration.Float%
18ratio-Activity at 18.3 nM% Activity of ratio channel at given concentration.Float%
19ratio-Activity at 40.9 nM% Activity of ratio channel at given concentration.Float%
20ratio-Activity at 91.5 nM% Activity of ratio channel at given concentration.Float%
21ratio-Activity at 205 nM% Activity of ratio channel at given concentration.Float%
22ratio-Activity at 457 nM% Activity of ratio channel at given concentration.Float%
23ratio-Activity at 1.02 uM% Activity of ratio channel at given concentration.Float%
24ratio-Activity at 2.29 uM% Activity of ratio channel at given concentration.Float%
25ratio-Activity at 5.11 uM% Activity of ratio channel at given concentration.Float%
26ratio-Activity at 11.4 uM% Activity of ratio channel at given concentration.Float%
27ratio-Activity at 25.6 uM% Activity of ratio channel at given concentration.Float%
28ratio-Activity at 57.1 uM% Activity of ratio channel at given concentration.Float%
29ikb-luc-Fit_LogAC50The logarithm of the AC50 from a fit of the ikb-luc data to the Hill equation.Float
30ikb-luc-Fit_HillSlopeThe Hill slope from a fit of the ikb-luc data to the Hill equation.Float
31ikb-luc-Fit_R2R^2 fit value of ikb-luc curve. Closer to 1.0 equates to better Hill equation fit.Float
32ikb-luc-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ikb-luc data to the Hill equation.Float%
33ikb-luc-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ikb-luc data to the Hill equation.Float%
34ikb-luc-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
35ikb-luc-Excluded PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
36ikb-luc-Activity at 0.70 nM% Activity of ikb-luc channel at given concentration.Float%
37ikb-luc-Activity at 1.6 nM% Activity of ikb-luc channel at given concentration.Float%
38ikb-luc-Activity at 3.7 nM% Activity of ikb-luc channel at given concentration.Float%
39ikb-luc-Activity at 8.2 nM% Activity of ikb-luc channel at given concentration.Float%
40ikb-luc-Activity at 18.3 nM% Activity of ikb-luc channel at given concentration.Float%
41ikb-luc-Activity at 40.9 nM% Activity of ikb-luc channel at given concentration.Float%
42ikb-luc-Activity at 91.5 nM% Activity of ikb-luc channel at given concentration.Float%
43ikb-luc-Activity at 205 nM% Activity of ikb-luc channel at given concentration.Float%
44ikb-luc-Activity at 457 nM% Activity of ikb-luc channel at given concentration.Float%
45ikb-luc-Activity at 1.02 uM% Activity of ikb-luc channel at given concentration.Float%
46ikb-luc-Activity at 2.29 uM% Activity of ikb-luc channel at given concentration.Float%
47ikb-luc-Activity at 5.11 uM% Activity of ikb-luc channel at given concentration.Float%
48ikb-luc-Activity at 11.4 uM% Activity of ikb-luc channel at given concentration.Float%
49ikb-luc-Activity at 25.6 uM% Activity of ikb-luc channel at given concentration.Float%
50ikb-luc-Activity at 57.1 uM% Activity of ikb-luc channel at given concentration.Float%
51luc-Fit_LogAC50The logarithm of the AC50 from a fit of the luc data to the Hill equation.Float
52luc-Fit_HillSlopeThe Hill slope from a fit of the luc data to the Hill equation.Float
53luc-Fit_R2R^2 fit value of luc curve. Closer to 1.0 equates to better Hill equation fit.Float
54luc-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the luc data to the Hill equation.Float%
55luc-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the luc data to the Hill equation.Float%
56luc-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
57luc-Excluded PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
58luc-Activity at 0.70 nM% Activity of luc channel at given concentration.Float%
59luc-Activity at 1.6 nM% Activity of luc channel at given concentration.Float%
60luc-Activity at 3.7 nM% Activity of luc channel at given concentration.Float%
61luc-Activity at 8.2 nM% Activity of luc channel at given concentration.Float%
62luc-Activity at 18.3 nM% Activity of luc channel at given concentration.Float%
63luc-Activity at 40.9 nM% Activity of luc channel at given concentration.Float%
64luc-Activity at 91.5 nM% Activity of luc channel at given concentration.Float%
65luc-Activity at 205 nM% Activity of luc channel at given concentration.Float%
66luc-Activity at 457 nM% Activity of luc channel at given concentration.Float%
67luc-Activity at 1.02 uM% Activity of luc channel at given concentration.Float%
68luc-Activity at 2.29 uM% Activity of luc channel at given concentration.Float%
69luc-Activity at 5.11 uM% Activity of luc channel at given concentration.Float%
70luc-Activity at 11.4 uM% Activity of luc channel at given concentration.Float%
71luc-Activity at 25.6 uM% Activity of luc channel at given concentration.Float%
72luc-Activity at 57.1 uM% Activity of luc channel at given concentration.Float%
73Compound TypeNCGC designation for compound stage: 'qHTS Exploratory', 'NIHSMR', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String
74Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

Data Table (Concise)
Classification
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