JNK-IN-8

JNK-IN-8 inhibits c-Jun phosphorylation in HeLa and A375 cells with EC50 of 486 nM and 338 nM, respectively. JNK-IN-8 shows a dramatic improvement in selectivity and eliminated binding to IRAK1, PIK3C3, PIP4K2C, and PIP5K3. JNK-IN-8 requires Cys116 for JNK2 inhibition. ^[1]

JNK-IN-8 (10 mM) suppresses the IL-1β-stimulated phosphorylation of c-Jun in IL-1R cells, an established substrate of the JNKs. JNK-IN-8 covalently attaches to the JNK isoforms caused a small retardation in the electrophoretic mobility of the JNK isoforms. ^[2]

JNK-IN-8 is discovered to inhibit JNK kinase by broad-based kinase selectivity profiling of a library of acrylamide kinase inhibitors based on the structure of imatinib using the KinomeScan approach. JNK-IN-8 possesses distinct regiochemistry of the 1,4-dianiline and 1,3-aminobenzoic acid substructures relative to imatinib and uses an N,N-dimethyl butenoic acetamide warhead to covalently target Cys154. JNK-IN-8 adopts an L-shaped type I binding conformation to access Cys 154 located toward the lip of the ATP-binding site. ^[3]

JNK-IN-8 is a potent JNK inhibitor that specially targets JNK activation. It has anti-fungal activity.

• 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.

• Animal Models

  C57BL/6 mice

• Dosages

  10 mg/kg

• Administration

  i.p.