JNK-IN-8

Alias: JNK Inhibitor XVI; JNK-IN 8; JNK-IN 8; JNK-IN8; c-Jun N-terminal Kinase Inhibitor XVI
Cat No.:V0472 Purity: ≥98%
JNK-IN-8 (JNK Inhibitor XVI; JNK-IN8) is the first irreversible/covalent inhibitor of pan-JNK (c-Jun N-terminal Kinase) with potential antitumor activity.
JNK-IN-8 Chemical Structure CAS No.: 1410880-22-6
Product category: JNK
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

JNK-IN-8 (JNK Inhibitor XVI; JNK-IN8) is the first pan-JNK (c-Jun N-terminal Kinase) inhibitor that is irreversible and covalent and has potential antitumor activity. By covalently attaching to Cys116 in their catalytic sites and inhibiting JNK1/2/4 with IC50 values of 4.7 nM, 18.7 nM, and 1 nM. For investigating JNK-dependent signal transduction, JNK-IN-8 is a helpful probe.

Biological Activity I Assay Protocols (From Reference)
Targets
JNK3 (IC50 = 1 nM); JNK1 (IC50 = 4.7 nM); JNK2 (IC50 = 18.7 nM); Kit (V559D,T670I) (IC50 = 56 nM)
ln Vitro
JNK-IN-8 has an EC50 of 486 nM and 338 nM, respectively, in HeLa and A375 cells where it prevents c-Jun phosphorylation. IRAK1, PIK3C3, PIP4K2C, and PIP5K3 no longer bind to JNK-IN-8, which results in a notable improvement in selectivity. For JNK2 inhibition, Cys116 is necessary in JNK-IN-8.[1]
JNK-IN-8 (10 mM) inhibits the JNKs' well-known substrate, IL-1R cells' induced phosphorylation of c-Jun. The covalent attachment of JNK-IN-8 to the JNK isoforms resulted in a slight reduction in the mobility of the JNK isoforms during electrophoresis.[2]
JNK-IN-8 was found to inhibit JNK kinase by using the KinomeScan method to profile a library of acrylamide kinase inhibitors based on the structure of imatinib. JNK-IN-8 uses an N,N-dimethyl butenoic acetamide warhead to covalently target Cys154 and differs from imatinib in its regiochemistry of the 1,4-dianiline and 1,3-aminobenzoic acid substructures. To reach Cys 154, which is close to the lip of the ATP-binding site, JNK-IN-8 adopts an L-shaped type I binding conformation. [3]
ln Vivo
JNK-IN-8 is a novel and potent JNK inhibitor that specially targets JNK activation. It has anti-fungal activity.
Enzyme Assay
Kinetics of binding assay Pretreatment of A375 cells with 1 μM JNK-IN-8 is carried out for the specified times. Remove the medium and give it three PBS washes. Re-suspend the cell pellet in 1 mL of lysis buffer, which contains phosphatase inhibitor cocktail, protease inhibitor cocktail, 1% NP-40, 1% CHAPS, 25 mM Tris, and 150 mM NaCl. Rotate in a circle for 30 minutes at 4 °C. Lysates are removed by centrifugation in the Eppendorf at 1.4×104 rpm for 15 min. The cleared lysates are gel filtered through 10DG columns into Kinase Buffer (0.1% NP-40, 20 mM HEPES, 150 mM NaCl, Phosphatase Inhibitor Cocktail, and Protease Inhibitor Cocktail). The gel-filtered lysate's total protein concentration should be between 5 and 15 mg/ml. The probe from ActivX is used to label cell lysate for 1 hour at a 5 μM concentration. In order to get rid of extra reagents and change the buffer, samples are reduced with DTT, and cysteines are blocked with iodoacetamide. Add 50 μL of streptavidin bead slurry and 1 vol of 2× Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2× PBS). Rotate end to end for 2 hours, and then centrifuge at 7,000 rpm for 2 minutes. Wash three times with PBS and three times with 1× Binding Buffer. Beads are given 30 μL 1× of sample buffer before being heated at 95°C for 10 minutes. Run samples at 110 volts through an SDS-PAGE gel.
Cell Assay
Interleukin Receptor 1 (IL1R)-expressing HEK-293 cells are grown in DMEM, which has been supplemented with 10% FBS, 2 mM glutamine, and 1 antimycotic/antibiotic solution. Before being treated with DMSO or JNK-IN-8 or being stimulated with 2 M anisomycin for 1 hour, cells are serum starved for 18 hours[1]. Lysates are then clarified by centrifugation for 10 minutes at 16000 g and 4°C.
Animal Protocol


References

[1]. Discovery of potent and selective covalent inhibitors of JNK. Chem Biol. 2012 Jan 27;19(1):140-54.

[2]. Developing irreversible inhibitors of the protein kinase cysteinome. Chem Biol. 2013 Feb 21;20(2):146-59.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C29H29N7O2
Molecular Weight
507.59
Exact Mass
507.24
Elemental Analysis
C, 68.62; H, 5.76; N, 19.32; O, 6.30
CAS #
1410880-22-6
Appearance
Solid powder
SMILES
CC1=C(C=CC(=C1)NC(=O)C2=CC(=CC=C2)NC(=O)/C=C/CN(C)C)NC3=NC=CC(=N3)C4=CN=CC=C4
InChi Key
GJFCSAPFHAXMSF-UXBLZVDNSA-N
InChi Code
InChI=1S/C29H29N7O2/c1-20-17-24(11-12-25(20)34-29-31-15-13-26(35-29)22-8-5-14-30-19-22)33-28(38)21-7-4-9-23(18-21)32-27(37)10-6-16-36(2)3/h4-15,17-19H,16H2,1-3H3,(H,32,37)(H,33,38)(H,31,34,35)/b10-6+
Chemical Name
3-[[(E)-4-(dimethylamino)but-2-enoyl]amino]-N-[3-methyl-4-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide
Synonyms
JNK Inhibitor XVI; JNK-IN 8; JNK-IN 8; JNK-IN8; c-Jun N-terminal Kinase Inhibitor XVI
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO: ~100 mg/mL (~197.0 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL
Solubility (In Vivo)
2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10mg/mL
 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 1.9701 mL 9.8505 mL 19.7009 mL
5 mM 0.3940 mL 1.9701 mL 3.9402 mL
10 mM 0.1970 mL 0.9850 mL 1.9701 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
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Biological Data
  • Chemical structures for JNK inhibitors. Chem Biol . 2012 Jan 27;19(1):140-54.
  • Mass spectra for THZ-IN-2 labeled JNK1 protein. Chem Biol . 2012 Jan 27;19(1):140-54.
  • Crystal structure of complex JNK-IN-2 and JNK-IN-7 with JNK3. Chem Biol . 2012 Jan 27;19(1):140-54.
  • Cellular pathway profiling with Western-blot. Chem Biol . 2012 Jan 27;19(1):140-54.
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