JNK-IN-7

Cas No.:	1408064-71-0
Chemical Name:	Jnk-IN-7
Synonyms:	JNK-IN-7;JNK inhibitor;(E)-3-(4-(dimethylamino)but-2-enamido)-N-(4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide;BDBM86632;BCP13495;Benzamide, 3-[[4-(dimethylamino)-1-oxo-2-buten-1-yl]amino]-N-[4-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-
SMILES:	O=C(C1C([H])=C([H])C([H])=C(C=1[H])N([H])C(/C(/[H])=C(\[H])/C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C1=NC([H])=C([H])C(C2=C([H])N=C([H])C([H])=C2[H])=N1
Formula:	C28H27N7O2
M.Wt:	493.5597
Purity:	>98%
Sotrage:	2 years -20°C Powder, 2 weeks 4°C in DMSO, 6 months -80°C in DMSO

Description:	JNK-IN-7 is a relatively selective JNKs inhibitor(IC50= 1.54/1.99/0.75 for JNK1/2/3); also bound to IRAK1, PIK3C3, PIP5K3 and PIP4K2C.
Target:	JNK3:0.7 nM (IC50) JNK1:1.5 nM (IC50) JNK2:2 nM (IC50)
In Vitro:	JNK-IN-7 is a relatively selective JNK inhibitor in cells. In addition to JNK 1, 2, 3, JNK-IN-7 also binds to IRAK1(IC50=14.1 nM), YSK4 (IC50=4.8 nM), ERK3 (IC50=22 nM), PIK3C3, PIP5K3 and PIP4K2C[1]. Expression of divalent metal-ion transporter 1 (DMT1) in HCT116 is demonstrated to be markedly decreased under stimulation with TNF for 24 and 48 h, while JNK-IN-7 can significantly reverse the decrease. TNF can down-regulate DMT1 expression, while JNK-IN-7 can markedly suppress this function[2].
Kinase Assay:	A375 cells are pre-treated with 1 μM JNK-IN-7 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). Rotate end-to-end for 30 min at 4°C. Lysates are 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 is labeled with the probe 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 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 is immunoblotted with JNK antibody[1].
Cell Assay:	Intestinal epithelial cell line (HCT116) is cultured in DMEM medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), Penicillin (100 U/mL) and Streptomycin (100 g/mL), 2 mM L-gentamycin, and 50 μM 2-ME. These cells are stimulated with TNF (20 ng/mL), LPS (100 ng/mL), and IFN-γ (20 ng/mL), respectively. After 24 or 48 h of culture, cells are harvested followed by extraction of total RNA, and the levels of DMT1 mRNA are analyzed by qRT-PCR. To determine the mechanisms of TNF involved in regulating DMT1 expression, JNK-IN-7 (1 μM), NF-κB inhibitor (BAY 11-7082, 1 μM), and caspase-3/8 inhibitor (Z-DEVD-FMK, 50 μM) are also added into the culture medium. After 48 h of culture, cells are then collected to detect the expression of DMT1 by qRT-PCR[2].
References:	[1]. Zhang T, et al. Discovery of Potent and Selective Covalent Inhibitors of JNK. Chem Biol. 2012 Jan 27;19(1):140-54. [2]. Wu W, et al. Divalent metal-ion transporter 1 is decreased in intestinal epithelial cells and contributes to the anemia in inflammatory bowel disease. Sci Rep. 2015 Nov 17;5:16344.