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BioAssay: AID 1284

Dose response biochemical screening assay for inhibitors of c-Jun N-Terminal Kinase 3 (JNK3)

The c-Jun N-Terminal Kinases (JNK) are members of the mitogen activated protein (MAP) kinase family of enzymes. Among the many substrates phosphorylated by JNKs is the N-terminal activation domain of the transcription factor c-Jun. So far, three genes have been found to encode for JNK kinases, namely JNK1, JNK2 and JNK3. Whereas JNK1 and JNK2 exhibit a ubiquitous human expression pattern, JNK3 more ..
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 Tested Compounds
 Tested Compounds
All(362)
 
 
Active(57)
 
 
Inactive(305)
 
 
 Tested Substances
 Tested Substances
All(362)
 
 
Active(57)
 
 
Inactive(305)
 
 
 Related BioAssays
 Related BioAssays
AID: 1284
Data Source: The Scripps Research Institute Molecular Screening Center (JNK3_INH_TR-FRET_1536_IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2008-05-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 57
Related Experiments
AIDNameTypeComment
746Primary biochemical high-throughput screening assay for inhibitors of the c-Jun N-Terminal Kinase 3 (JNK3)Screeningdepositor-specified cross reference
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Scripps Florida
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number ML00111
Grant Proposal PI: Philip LoGrasso

External Assay ID: JNK3_INH_TR-FRET_1536_IC50

Name: Dose response biochemical screening assay for inhibitors of c-Jun N-Terminal Kinase 3 (JNK3)

Description:

The c-Jun N-Terminal Kinases (JNK) are members of the mitogen activated protein (MAP) kinase family of enzymes. Among the many substrates phosphorylated by JNKs is the N-terminal activation domain of the transcription factor c-Jun. So far, three genes have been found to encode for JNK kinases, namely JNK1, JNK2 and JNK3. Whereas JNK1 and JNK2 exhibit a ubiquitous human expression pattern, JNK3 expression appears to be largely restricted to the brain [1]. JNKs are activated by a variety of stimuli including cytokines, ultra violet light, growth factors and oxidative stress. It has been demonstrated that JNK3 plays a key role in Parkinson's disease (PD), a common neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons particular to the mid brain or substantia nigra. PD patients exhibit activation of c-Jun in their dopaminergic neurons. Consistently, this activation has also been observed in a common mouse model of PD consisting of mice treated with 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP) [2]. The examination of JNK deficient mice showed that both JNK2 and JNK3, but not JNK1, are required for MPTP-induced c-Jun activation and dopaminergic cell demise. Cyclooxygenase-2 (COX-2), a downstream target of JNK activation, has also been associated with dopaminergic cell death [3]. Studies involving a specific JNK inhibitor, SP600125, have validated this concept by showing a protective effect in a mouse model for Parkinson's Disease [4]. Based on those observations, the discovery of novel inhibitors of JNK3 may provide valuable probes to better understand the implication of this kinase in neurodegenerative diseases.

References:
1. Harper SJ, LoGrasso P. Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38. Cell Signal. 2001 May;13(5):299-310.
2. Willesen MG, Gammeltoft S, Vaudano E. Activation of the c-Jun N terminal kinase pathway in an animal model of Parkinson's disease. Ann N Y Acad Sci. 2002 Nov;973:237-40.
3.Hunot S, Vila M, Teismann P, Davis RJ, Hirsch EC, Przedborski S, Rakic P, Flavell RA. JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):665-70. Epub 2004 Jan 2.
4.Xia XG, Harding T, Weller M, Bieneman A, Uney JB, Schulz JB. 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. 2001 Aug 28;98(18):10433-8. Epub 2001 Aug 14.

Keywords:
JNK3, MPTP, SP600125, c-Jun N-terminal kinase, Phospho-c-jun, Dopaminergic neurons, Parkinson, MAPK10, Mitogen-activated protein kinase 10, p493F12, p54bSAPK, PRKM10, time-resolved fluorescence energy transfer, TR-FRET, FRET, HTRF, dose response, 1536, Scripps, Scripps Florida, Molecular Library Screening Center Network, MLSCN.
Protocol
Assay Overview:
The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary biochemical high-throughput screening assay for inhibitors of the c-Jun N-Terminal Kinase 3 (JNK3)," (PubChem AID 746). In this assay phosphorylation of the JNK3 substrate Activating Transcription Factor 2 (ATF2) is measured using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). The assay uses biotinylated ATF2 (b-ATF2), which binds both XL-665-labeled streptavidin (SA-XL-665) and a europium-labeled antibody (Eu-Ab) that specifically recognizes the phosphorylated form of b-ATF2. Upon b-ATF2 phosphorylation by JNK3, Eu-Ab binds the phosphorylated b-ATF2. The addition of SA-XL-665 brings the FRET donor (Eu-Ab) and acceptor (SA XL-665) pair into close proximity, allowing FRET to occur. As designed, a compound that inhibits JNK3 activity will prevent JNK3-mediated phosphorylation of b-ATF, Eu-Ab binding to b-ATF2, therefore reducing FRET. Compounds were tested in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 93 micromolar.
Protocol Summary:
Prior to the start of the assay, 4 microliters of a solution containing 1.25 micromolar ATP and 250 nM b-ATF2 peptide (substrate) in 2X assay buffer (100 mM HEPES pH, 20 M MgCl2, 2 mM DTT, 0.1% BSA, pH 7.4) were dispensed into 1536 microtiter plates. Next, 47 nL of test compound, High Control compound (JNK3 selective inhibitor SP600125; 10 micromolar final nominal concentration), or Low Control compound DMSO; final concentration 0.93 %) were added to the appropriate wells. Then, the assay was started by dispensing 1 ul of 0.625 nM activated JNK3 in 2X assay buffer. After 30 minutes of incubation at room temperature (RT), 5 ul of 2X Quenching Buffer (100 mM HEPES, 0.22 uM SA-XL 665, 0.043 ng/ul Eu-Ab, 50 mM EDTA, 400 mM KF, pH 7.25) was added to each well. Next, the plates were incubated for 1 hour at RT and time-resolved fluorescence emission was read at 618 nm and 671 nm for 30 seconds on a microplate reader (Viewlux, Perkin-Elmer) upon excitation at 340 nm.
To normalize data, fluorescence emission values measured from both wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = (I671 nm / I618 nm) x 10,000
where I = measured fluorescence emission intensity at the enumerated wavelength in nanometers
The percent inhibition for each compound was calculated as follows:
% Inhibition = ((Ratio Test_Compound - Median Ratio Low_Control) / (Median Ratio High_Control - Median Ratio Low_Control))*100
Where:
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing SP600125.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for the X-intercept at the 50% inhibition level of the Y-intercept. In cases where the highest concentration tested did not result in > 50% inhibition or where no curve fit was achieved, the IC50 was determined manually depending on the observed inhibition at the individual concentrations. Compounds with IC50 values greater than 10 micromolar were considered inactive. Compounds with IC50 values equal to or less than 10 micromolar were considered active.
Any compound with a percent inhibition value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent inhibition value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by potency, with the most potent compounds assigned the highest activity scores.
List of reagents:
EDTA (Sigma-Aldrich, part E7889)
ATP (Sigma-Aldrich, part A6559)
HEPES pH 7.3 (Invitrogen, part 15630-114)
MgCl2 (Sigma-Aldrich, part M4880-100g)
BSA (Sigma-Aldrich, part A3803-100g)
bATF2 peptide (synthesized in-house)
activated JNK3 enzyme (expressed in-house and activated with mitogen-activated protein kinase kinase 7)
SA-XL 665 (Cisbio, part 611SAXLB)
Eu-Ab to b-ATF2 (Cisbio, part 61P12KAZ)
1536-well plates (Greiner, part 789176)
Comment
Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
The inactive compounds of this assay have activity score range of 0 to 41 and active compounds range of activity score is 41 to 100.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the antagonist assay in M concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Float
11Inhibition at 4.7 nM (0.0047μM**)Value of %inhibition at 4.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 14.2 nM (0.0142μM**)Value of %inhibition at 14.2 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 42.6 nM (0.0426μM**)Value of %inhibition at 42.6 nanomolar inhibitor concentration; average of triplicate measurement. Float%
14Inhibition at 127.7 nM (0.1277μM**)Value of %inhibition at 127.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 383.2 nM (0.3832μM**)Value of %inhibition at 383.2 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 1.1 uM (1.1μM**)Value of %inhibition at 1.1 micromolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 3.4 uM (3.4μM**)Value of %inhibition at 3.4 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 10.4 uM (10.4μM**)Value of %inhibition at 10.4 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 31.0 uM (31μM**)Value of %inhibition at 31.0 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 93.1 uM (93.1μM**)Value of %inhibition at 93.1 micromolar inhibitor concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.

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