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 ..
BioActive Compounds: 57
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)
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 . 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) . 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 . Studies involving a specific JNK inhibitor, SP600125, have validated this concept by showing a protective effect in a mouse model for Parkinson's Disease . 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.
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.
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.
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.
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
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)
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)