All solvents and reagents were used as obtained. 1H NMR spectra were recorded with a Varian Inova 600 NMR spectrometer and referenced to dimethylsulfoxide. Chemical shifts are expressed in ppm. Mass spectra were measured with Waters Micromass ZQ using an ESI source coupled to a Waters 2525 HPLC system operating in reverse mode with a Waters Sunfire C18 5 μm, 4.6 mm x 50 mm column. Purification of compounds was performed with either a Teledyne ISCO CombiFlash Rf system or a Waters Micromass ZQ preparative system. The purity was analyzed on an above-mentioned Waters LC-MS Symmetry (C18 column,4.6 mm x 50 mm, 5 μM) using a gradient of 5-95% methanol in water containing 0.05% trifluoacetic acid (TFA). Detailed synthetic schemes and characterization data are presented in the supplementary data
JNK1/2/3 specific assay condition (Cascade format) – MAPK8 (JNK1)
The 2X MAPK8 (JNK1)/inactive MAPKAPK3/Ser/Thr 04 Peptide Mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT. The final 10 μL kinase reaction consists of 3.3-13.3 ng MAPK8 (JNK1), 20 ng inactive MAPKAPK3, and 2 μM Ser/Thr 04 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT. After the 1 hour kinase reaction incubation, 5 μL of a 1:1024 dilution of development reagent A is added. MAPK9 (JNK2) The 2X MAPK9 (JNK2)/inactive MAPKAPK3/Ser/Thr 04 peptide mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT. The final 10 μL kinase reaction consists of 2.4 - 9.8 ng MAPK9 (JNK2), 20 ng inactive MAPKAPK3 and 2 μM Ser/Thr 04 peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT. After 1 hour kinase reaction incubation, 5 μL of a 1:1024 dilution of development reagent A is added. MAPK10 (JNK3) The 2X MAPK10 (JNK3)/inactive MAPKAPK3/Ser/Thr 04 peptide mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT. The final 10 μL kinase reaction consists of 1.3-5.3 ng MAPK10 (JNK3), 20 ng inactive MAPKAPK3 and 2 μM Ser/Thr 04 peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT. After the 1 hour kinase reaction incubation, 5 μL of a 1:1024 dilution of development reagent A is added.
Intact protein analysis
For each analysis, ~100 pmol JNK protein +/− inhibitor (JNK-IN-2 or JNK-IN-7) was injected onto a self-packed reversed phase column (1/32” O.D. x 500 μm I.D., 5 cm of POROS 10R2 resin). After desalting, protein was eluted with an HPLC gradient (0-100% B in 4 minutes, A=0.2M acetic acid in water, B=0.2 M acetic acid in acetonitrile, flow rate = 10 μL/min) into a QTRAP mass spectrometer (AB Sciex, Toronto, Canada) or an LTQ Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA). The QTRAP was operated in Q1 MS mode at unit resolution scanning at 2000 amu/sec. LTQ OrbitrapMS spectra were acquired in centroid mode using the electron multipliers for ion detection. Mass spectra were deconvoluted using MagTran1.03b2 software (Zhang et al, 1998
Protease digestion and nanoLC/MS analysis of peptide fragments
JNK-IN-2 or JNK-IN-7 treated JNK (25 μg, ~620 pmol) was diluted with ammonium bicarbonate buffer, pH 8.0 then reduced for 30 min at 56 °C with 10 mM DTT. After cooling for 5 min, the protein was alkylated with 22.5 mM iodoacetamide for 30 min at room temperature in the dark, and digested overnight with 1.5 μg of trypsin at 37 °C. In the morning, 1 μg of Glu-C was added, and the solution further incubated at 37 °C for 8 hr. Digested peptides (~2 pmol) were injected onto a self-packed pre-column (4 cm POROS10R2) and eluted into the mass spectrometer (LTQ OrbitrapVelos, ThermoFisher Scientific). Peptides were subjected to MS2 by CAD (electron multiplier detection, relative collision energy 35%, q = 0.25) as well as HCD (image current detection, resolution @ m/z 400 = 7500, relative collision energy 35%).
Cell-Based Assays for c-Jun Phosphorylation
The cell based kinase assays for c-Jun phosphorylation carried out by using the LanthaScreen™ c-Jun (1–79) HeLa cell line (Life Technologies, Carlsbad, CA) which stably express GFP-c-Jun 1–79 and GFP-ATF2 19–106, respectively. Phosphorylation was determined by measuring the time resolved FRET (TR-FRET) between a terbium labeled phospho-c-Jun specific antibody and GFP (22). The cells were plated in white tissue culture treated 384 well plates at a density of 10,000 cell per well in 32 μL assay medium (Opti-MEM®, supplemented with 0.5% charcoal/dextran-treated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, 0.1mM nonessential amino acids, 1mM sodium pyruvate, 25 mM Hepes, pH 7.3, and lacking phenol red). After overnight incubation, cells were pretreated for 90 min with compound (at indicated concentration) diluted in 4 μL assay buffer followed by 30 min of stimulation with 5 ng/ml of TNF-α in 4 μL assay buffer (final assay volume was 40 μl). The medium was then removed by aspiration and the cells were lysed by adding 20 μl of lysis buffer (20 mM Tris-HCl, pH7.6, 5 mM EDTA, 1% Nonidet P-40 substitute, 5 mM NaF, 150 mM NaCl, and 1:100 protease and phosphatase inhibitor mix, SIGMA P8340 and P2850, respectively). The lysis buffer included 2 nM of the terbium-labeled anti-c-Jun (pSer73) detection antibodies (Life Technologies). After allowing the assay to equilibrate for 60 minutes at room temperature, TR-FRET emission ratios were determined on a BMG Pherastar fluorescence plate reader (BMG Labtech, Cary, NC) using the following parameters: excitation at 340 nm, emission 520 nm and 490 nm; 100 μs lag time; 200 μs integration time; emission ratio = Em 520 / Em 490. All data were analyzed and plotted using Graphpad Prism 4.
High Throughput Microscopy
Cells were plated at 7500 cells/well in 96-well microscopy plates (Corning) in recommended media for 24 hours, and then starved in media lacking serum for 16 hours. Cells were pre-treated for 180 minutes with 10-fold stock solutions of JNK inhibitors and for 10 min with control compounds MK2206 (allosteric Akt inhibitor;, Haoyuan Chemexpress co. limited; Hirai et al., 2010
), PD0325901 (allosteric Mek inhibitor; Haoyuan Chemexpress co. limited; Barrett et al., 2008
), SB239063 (ATP-competitive p38 inhibitor; Haoyuan Chemexpress co. limited; Underwood et al., 2000
), KIN001-040 (ATP-competitive JAK1,2,3 inhibitor; Haoyuan Chemexpress co. limited;. Thompson et al., 2002
) and KIN001-208 (IKK inhibitor VIII, Haoyuan Chemexpress co. limited., Murata et al., 2004
) and treated with 10- fold stock solutions of IGF-1, IL-6, TNF-α (all PeproTech) or anisomycin for 60 minutes. Cells were fixed in 2% paraformaldehyde for 10 min at room temperature and washed with PBS-T (Phosphate Buffered Saline, 0.1% Tween 20). Cells were permeabilized in methanol for 10 min at room temperature, washed with PBS-T, and blocked in Odyssey Blocking Buffer (LI-COR Biosciences) for 1 hour at room temperature. Cells were incubated overnight at 4°C with antibody specific for Erk1/2(pT202/pY204), Akt(pS473), cJUN(pS73), pP38(T180/Y182) and pSTAT3(Y705) (Cell Signaling Technology), pRSK1(S380) and pMSK1(S376) (Epitomics) and NF-κB (Santa Cruz Biotechnology) diluted 1:400 in Odyssey Blocking Buffer. Cells were washed three times in PBS-T and incubated with rabbit-specific secondary antibody labeled with Alexa Fluor 647 (Invitrogen) diluted 1:2000 in Odyssey Blocking Buffer. Cells were washed once in PBS-T, once in PBS and incubated in 250 ng/ml Hoechst 33342 (Invitrogen) and 1:1000 Whole Cell Stain (blue; Thermo Scientific) solution. Cells were washed two times with PBS and imaged in an imageWoRx high-throughput microscope (Applied Precision). Data was plotted using DataPflex (Hendriks et al., 2010
Binding Kinetics assay
A375 cells (ATCC® CRL-1619™) were pre-treated with 1μM compound for the indicated amounts of time. Remove the medium and wash 3 times with PBS. Resuspend the cell pellet with 1 mL Lysis Buffer (1% NP-40, 1% CHAPS, 25 mM Tris, 150 mM NaCl, Phosphatase Inhibitor Cocktail, Roche 04906845001, and Protease Inhibitor Cocktail, Roche 11836170001). Rotate end-to-end for 30 min at 4°C. Lysates were cleared by centrifugation at 14000 rpm for 15 min in the Eppendorf. The cleared lysates gel filtered into Kinase Buffer (0.1% NP-40, 20 mM HEPES, 150 mM NaCl, Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail) using Bio-Rad 10DG colums. The total protein concentration of the gel-filtered lysate should be around 5-15 mg/ml. Cell lysate was labeled with the probe from ActivX® at 5 μM for 1 hour. Samples were reduced with DTT, and cysteines were blocked with iodoacetamide and gel filtered to remove excess reagents and exchange the buffer. Add 1 volume of 2X Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2X PBS) and 50 μL streptavidin bead slurry and rotate end-to-end for 2 hours, centrifuge at 7000 rpm for 2 min. Wash 3 times with 1X Binding Buffer and 3 times with PBS. Add 30 μL 1X sample buffer to beads, heat samples at 95°C for 10 min. Run samples on an SDS-PAGE gel at 110V. After transferred, the membrane was immunoblotted with JNK antibody (Cell signaling 9258).
Incubate 1 μM JNK-IN-5 with purified JNK3 protein for indicated time period, then add the ATP-Biotin probe from ActivX® at 5 μM for 10 min. Denature the protein by adding same volume 8 M urea solution and gel filtered to remove excess reagents and exchange the buffer. Add 1 volume of 2X Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2X PBS) and 50 μL streptavidin bead slurry and rotate end-to-end for 2 hours, centrifuge at 7000 rpm for 2 min. Wash 3 times with 1X Binding Buffer and 3 times with PBS. Add 30 μL 1X sample buffer to beads, heat samples at 95°C for 10 min. Run samples on an SDS-PAGE gel at 110V. After transferred, the membrane was immunoblotted with JNK antibody (Cell signaling 9252).
Lysis Buffer contained 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (w/v) 1 mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM Benzamidine and 2 mM phenylmethanesulphonylfluoride (PMSF) and supplemented with 1% (v/v) Triton X-100. Kinase assay buffer contained 50 mM Tris/HCl, pH 7.5 and 0.1 mM EGTA.
Cell culture, treatments and cell lysis
HEK-293 cells stably expressing Interleukin Receptor 1 (HEK293-IL1R) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and 1×antimycotic/antibiotic solution. Cells were serum starved for 18h before incubation with DMSO or different inhibitors, stimulated with 2 μM anisomycin (Sigma) for 1h and lysates were clarified by centrifugation for 10 min at 16000 g and 4°C.
Rabbit polyclonal antibodies against total pan JNK isoforms ((#9252), phospho-pan JNK isoforms (Thr183/Tyr185), (#4668), total p38 (#9212) or phospho-p38 MAPK (Thr180/Tyr182), (4631 resp.), total c-Jun (#9165), phospho-c-Jun (Ser63), (#9261) and phospho-MSK1 (Ser376) (#9591) were from Cell Signalling technology.
SDS-PAGE and western blot
Cell lysates (30 μg) were resolved by electrophoresis on SDS polyacrylamide gels (10%) or Novex 4-12% gradient gels, and electroblotted to nitrocellulose membranes. Membranes were blocked with 5% skimmed milk (w/v) in 50 mM Tris/HCl, pH 7.5, 0.15 M NaCl and 0.1% (v/v) Tween (TBST Buffer). Primary antibodies were used at a concentration of 1 μg/ml, diluted in 5% skimmed milk in TBST and incubated overnight at 4°C. Detection of immune-complexes was performed using horseradish-peroxidase-conjugated secondary antibodies (Pierce) and an enhanced-chemiluminescence reagent (in-house).
JNK2 Kinase assays
Wild type JNK2 or mutant JNK2[Cys116Ser] was activated in a reaction mixture containing 2 μM JNK2, 200 nM MKK4, 200 nM MKK7 in kinase assay buffer containing 0.1 mM ATP and 10 mM magnesium chloride. After incubation at 30 min at 30°C the reaction mixture was snap frozen in aliquots. Activity of JNK2 was assessed in a total reaction volume of 50 μl containing 200 nM activated wild type JNK or mutant JNK2[Cys116Ser], in kinase buffer containing 0.1 mM [γ-32P]ATP (~500-1000 cpm/pmol), 10 mM magnesium chloride and 2 μM ATF2 (residues 19-96) as a substrate. The different inhibitors, or equivalent DMSO volume in controls, were added immediately before to the ATP. Reactions were terminated by adding 20 mM EDTA after 30 min at 30°C incubation 40 μl of the reaction mixture was applied to P81 phosphocellulose paper which were washed in 50 mM phosphoric acid and phosphorylated ATF2 peptide bound to p81 paper quantified by Cerenkov counting.