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BioAssay: AID 530

JNK3 AlphaScreen Assay

The c-jun N-terminal kinase (JNK) family are serine/threonine protein kinases that phosphorylate c-jun, a component of the transcription factor protein-1 (AP-1). JNK kinases are members of the mitogen-activated protein kinase family including the extracellular regulated kinases and p38 kinases. Three JNK genes (JNK 1, 2, and 3) have been identified in humans so far. JNK1 and JNK2 have a broad distribution and JNK3 is primarily found in neuronal tissues and cardiac myocytes. ..more
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 Tested Compounds
 Tested Compounds
All(10010)
 
 
Active(34)
 
 
Inactive(9062)
 
 
Inconclusive(1135)
 
 
 Tested Substances
 Tested Substances
All(11014)
 
 
Active(36)
 
 
Inactive(9788)
 
 
Inconclusive(1190)
 
 
 Related BioAssays
 Related BioAssays
AID: 530
Data Source: NCGC (ncgca-jnk3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2006-11-13
Modify Date: 2006-11-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 34
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

NCGC Assay Overview:

The c-jun N-terminal kinase (JNK) family are serine/threonine protein kinases that phosphorylate c-jun, a component of the transcription factor protein-1 (AP-1). JNK kinases are members of the mitogen-activated protein kinase family including the extracellular regulated kinases and p38 kinases. Three JNK genes (JNK 1, 2, and 3) have been identified in humans so far. JNK1 and JNK2 have a broad distribution and JNK3 is primarily found in neuronal tissues and cardiac myocytes.

JNK3 has been under consideration as a target for the treatment of neurodegenerative disorders. Inhibition of JNK3 has displayed neuroprotective actions on transient brain ischemia and reperfusion-induced neuronal death in the rat hippocampal CA1 region (Yang et al., 1997; Herdegen et al., 1998). JNK pathway is also involved in pathological process in a mouse model of Parkinson's disease (Xia et al., 2001).

Given these potential tissue distributions and links to neuroprotection, JNK3 inhibitors should provide useful investigational tools to study the role of this kinase in several diseases including, arthritis, stroke, myocardial infarction, ischemic injury and Parkinson's disease.

A homogeneous AlphaScreen is developed to measure the activity of JNK3. AlphaScreen is a two bead-based proximity-dependent chemical energy transfer luminescent assay platform. The phosphorylation of a biotinylated c-jun substrate by JNK3 can be detected with an antibody raised against phosphorylated c-jun. This process brings streptavidin donor beads and Protein-A acceptor beads to within a proximity range of <200 nm, and subsequently lead a chemical luminescent reaction upon exciting donor beads with 680 nm laser excitation light. Inhibition of JNK3 activity by small molecules and subsequent decrease in the antibody binding to biotinylated c-jun could attenuate or abolish this chemical luminescent reaction.

References:

Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, Rakic P and Flavell RA (1997) Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 389:865-870. [PMID: 9349820]

Herdegen T, Claret FX, Kallunki T, Martin-Villalba A, Winter C, Hunter T, and Karin M (1998) Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J.Neurosci. 18:5124-5135. [PMID: 9651196]

Xia XG, Harding T, Weller M, Bieneman A, Uney JB and Schulz JB (2001) Gene transfer of the JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson's disease. Proc.Natl.Acad.Sci.U.S.A. 98:10433-10438. [PMID: 11504916]
Protocol
NCGC Assay Protocol Summary:
GST-cJun (1-79) (Stratagene, La Jolla CA, #205145) was biotnylated by PerkinElmer (PerkinElmer-BioSignalInc. Montreal, Canada) and was detectable by the streptavidin Donor beads. Anti-(phospho)cJun (Ser73) antibody was from UpState (Charlottesvilles, VA, #06-659). JNK3-containing lysates were generated from HepG2 cells and pre-activated by interleukin1-beta and tumor necrosis factor alpha. All reagents were stored at -20 C before using. Phosphorylated biotinylated-GST-cJun was recognized by Anti-(phospho)cJun (Ser73) antibody and this process brings acceptor and donor beads together. Therefore JNK3 phosphorylation of cJun can be measured by Alpha reading by EnVision (PerkinElmer, Boston, MA).
The composition of kinase buffer was (in mM) 20 tris- HCl (pH 7.6) 10 MgCl2, 1 DTT, and 0.1 Na3VO4 and 0.01% Tween-20. Beads were diluted in detection buffer containing (in mM) 20 tris-HCl (pH 7.7), 20 NaCl and 80 EDTA and 0.3% BSA. The assay was performed at room temperature. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format (white solid bottom and untreated) as follows:


Sequence,Parameter,Value,Description
1,Reagent,3 uL,Biotin-GST-cJun, Anti-(phospho)cJun and pre-activated JNK3-containing lysates
2,Compound,23 nL,Final concentration was 0.6nM to 46uM
3,Time,15 minutes,Room Temperature
4,Reagent,1 uL,ATP final concentration 10uM
5,Time,2 hour,Room temp
6,Reagent,0.5 uL,2ng/well bead mixture
7,Time,3 hour,Room temp
8,Detector,EX 680 nm / singlet O2 / EM 520-620 nm,Envision (PerkinElmer, detected through aperture 1536A1)

Keywords: cJun, JNK3, AlphaScreen, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
Comment
Compound Ranking:

1. Compounds are first classified as being probable inhibitors (compounds interfering with the alpha screen), activators (compounds causing an increase in alpha screen readout), inactive, or inconclusive.

2. Within the inhibitors, 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: inhibitor, activator, inactive, or inconclusive.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
3EfficacyMaximal 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.String
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
6JNK3-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7JNK3-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8JNK3-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9JNK3-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float
10JNK3-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float
11JNK3-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12JNK3_ExcludedPointsWhich 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
13JNK3-Activity at 0.7 nM% Activity at given concentration.Float%
14JNK3-Activity at 1.6 nM% Activity at given concentration.Float%
15JNK3-Activity at 3.7 nM% Activity at given concentration.Float%
16JNK3-Activity at 8.2 nM% Activity at given concentration.Float%
17JNK3-Activity at 18.3 nM% Activity at given concentration.Float%
18JNK3-Activity at 41 nM% Activity at given concentration.Float%
19JNK3-Activity at 92 nM% Activity at given concentration.Float%
20JNK3-Activity at 205 nM% Activity at given concentration.Float%
21JNK3-Activity at 457 nM% Activity at given concentration.Float%
22JNK3-Activity at 1.02 uM% Activity at given concentration.Float%
23JNK3-Activity at 2.29 uM% Activity at given concentration.Float%
24JNK3-Activity at 5.11 uM% Activity at given concentration.Float%
25JNK3-Activity at 11.4 uM% Activity at given concentration.Float%
26JNK3-Activity at 25.6 uM% Activity at given concentration.Float%
27JNK3-Activity at 57.1 uM% Activity at given concentration.Float%
28Compound TypeNCGC designation for compound stage: 'qHTS Exploratory', 'NIHSMR', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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