Home MAPK JNK inhibitor JNK-IN-8

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JNK-IN-8 JNK inhibitor

Cat.No.S4901

JNK-IN-8 (JNK Inhibitor XVI) is the first irreversible JNK inhibitor for JNK1, JNK2 and JNK3 with IC50 of 4.7 nM, 18.7 nM and 1 nM, >10-fold selectivity against MNK2, Fms and no inhibition to c-Kit, Met, PDGFRβin A375 cell line.
JNK-IN-8 JNK inhibitor Chemical Structure

Chemical Structure

Molecular Weight: 507.59

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Quality Control

Batch: Purity: 99.16%
99.16

Cell Culture, Treatment & Working Concentration

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
human HeLa cells Function assay 1 h Inhibition of JNK3-mediated c-jun phosphorylation in human HeLa cells after 1 hr incubation, IC50=0.486 μM 25415535
human A375 cells Function assay 1 h Inhibition of JNK3-mediated c-jun phosphorylation in human A375 cells after 1 hr incubation, IC50=0.338 μM 25415535
Click to View More Cell Line Experimental Data

Solubility

In vitro
Batch:

DMSO : 100 mg/mL (197.0 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Water : Insoluble

Ethanol : Insoluble

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In vivo
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Chemical Information, Storage & Stability

Molecular Weight 507.59 Formula

C29H29N7O2

 Storage (From the date of receipt)
CAS No. 1410880-22-6 Download SDF Storage of Stock Solutions

Synonyms JNK Inhibitor XVI Smiles CC1=C(C=CC(=C1)NC(=O)C2=CC(=CC=C2)NC(=O)C=CCN(C)C)NC3=NC=CC(=N3)C4=CN=CC=C4

Mechanism of Action

Features
JNK-IN-8 and JNK-IN-7 are structurally very similar, but whereas the former is a specific covalent inhibitor of JNKs.
Targets/IC50/Ki
JNK3
(A375 cells)
1 nM
JNK1
(A375 cells)
4.7 nM
JNK2
(A375 cells)
18.7 nM
Kit (V559D,T670I)
(A375 cells)
56 nM
Kit (V559D)
(A375 cells)
92 nM
In vitro

JNK-IN-8 inhibits c-Jun phosphorylation in HeLa and A375 cells with EC50 of 486 nM and 338 nM, respectively. This compound shows a dramatic improvement in selectivity and eliminated binding to IRAK1, PIK3C3, PIP4K2C, and PIP5K3. It requires Cys116 for JNK2 inhibition.

This chemical (10 mM) suppresses the IL-1β-stimulated phosphorylation of c-Jun in IL-1R cells, an established substrate of the JNKs. It covalently attaches to the JNK isoforms caused a small retardation in the electrophoretic mobility of the JNK isoforms.

It is discovered to inhibit JNK kinase by broad-based kinase selectivity profiling of a library of acrylamide kinase inhibitors based on the structure of using the KinomeScan approach. This compound possesses distinct regiochemistry of the 1,4-dianiline and 1,3-aminobenzoic acid substructures. It adopts an L-shaped type I binding conformation to access Cys 154 located toward the lip of the ATP-binding site.

Kinase Assay
Binding Kinetics Assay
Binding kinetics assay A375 cells are pretreated with 1 μM JNK-IN-8 for the indicated amounts of time. Remove the medium and wash three 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, and Protease Inhibitor Cocktail). Rotate end to end for 30 min at 4℃. Lysates are cleared by centrifugation at 1.4×104 rpm for 15 min in the Eppendorf. The cleared lysates are gel filtered into Kinase Buffer (0.1% NP-40, 20 mM HEPES, 150 mM NaCl, Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail) using 10DG columns. The total protein concentration of the gel-filtered lysate should be around 5–15 mg/ml. Cell lysate is labeled with the probe from ActivX at 5 μM for 1 hour. Samples are reduced with DTT, and cysteines are blocked with iodoacetamide and gel filtered to remove excess reagents and exchange the buffer. Add 1 vol of 2× Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2× PBS) and 50 μL streptavidin bead slurry, and rotate end to end for 2 hours, centrifuge at 7,000 rpm for 2 min. Wash three times with 1× Binding Buffer and three times with PBS. Add 30 μL 1× sample buffer to beads; heat samples at 95℃ for 10 min. Run samples on an SDS-PAGE gel at 110V. After transferred, the membrane is immunoblotted with JNK antibody.
In vivo

JNK-IN-8 is a potent JNK inhibitor that specially targets JNK activation. It has anti-fungal activity.
References
  • [4] https://pubmed.ncbi.nlm.nih.gov/28112734/

Applications

Methods Biomarkers Images PMID
Western blot Notch1 / c-Myc / Cyclin D1 / p21 c-Jun / p-c-Jun / ATF2 / p-ATF2 / Elk-1 / pElk-1
S4901-WB2
27941886
Growth inhibition assay Cell viability
S4901-viability1
25847947

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