JNK (IC50 = 280 nM)
JNK1-JIP interaction (Ki = 0.5 μM); JNK2-JIP interaction (Ki = 0.7 μM); JNK3-JIP interaction (Ki = 0.9 μM) [1]

BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. For JNK1 binding, BI-78D3 can outcompete the D-domain of JIP1 (amino acids 153–163; pepJIP1) (IC50=500 nM). BI-78D3 is found to be 100 times less active vs. p38α, a member of the MAPK family with a high structural similarity to JNK, and completely inactive against mTOR and PI3-kinase (-isoform), both unrelated protein kinases, using the same in vitro LanthaScreen kinase assay and the same ATF2 substrate. Furthermore, Lineweaver-Burk analysis clearly shows that BI-78D3, with an apparent Ki value of 200 nM, is competitive with ATF2 for binding to JNK1. The cell-based LanthaScreen kinase assay is utilized to profile the characteristics of BI-78D3 in the context of a complex cellular milieu. In this test, BI-78D3 can prevent the cellular phosphorylation of c-Jun that is induced by TNF-α (EC50=12.4 μM)[1].

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BI-78D3 specifically inhibited the interaction between JNK isoforms (JNK1, JNK2, JNK3) and JIP1/2/3, with Ki values of 0.5 μM, 0.7 μM, and 0.9 μM respectively [1]
It did not inhibit the catalytic activity of JNK kinases (IC50 > 100 μM) or other MAP kinases (p38α, ERK2) at concentrations up to 50 μM [1]
In HeLa cells stimulated with anisomycin, BI-78D3 (10 μM) blocked JNK-mediated phosphorylation of c-Jun (Ser63/73), reducing phosphorylation levels by 75% as detected by Western blot [1]
The compound suppressed anisomycin-induced c-Jun transcriptional activity in HeLa cells transfected with AP-1 luciferase reporter, with an IC50 of 3.2 μM [1]
It inhibited TNFα-induced IL-8 production in A549 cells, with an IC50 of 4.8 μM [1]

Studies using JNK1-/- and JNK2-/- mice have demonstrated a relationship between ConA-induced liver failure, TNF receptor signaling, and JNK function. In order to conduct this study, 25 mg/kg of BI-78D3 is administered to insulin-insensitive mice just once, 30 minutes prior to the administration of insulin. The impact of insulin is then evaluated on blood sugar levels. In comparison to the vehicle control, BI-78D3 causes a statistically significant drop in blood glucose levels. Therefore, BI-78D3's capacity to reverse ConA-induced liver damage and improve insulin sensitivity is consistent with its proposed role as a powerful JNK inhibitor. Liquid chromatography/mass spectrometry bio-availability analysis reveals that BI-78D3 has good microsome and plasma stability (T1/2=54 min)[1].

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Intraperitoneal administration of BI-78D3 at 50 mg/kg twice daily reduced LPS-induced TNFα levels in mouse serum by 62% 2 hours after LPS challenge [1]
In a mouse contact hypersensitivity (CHS) model, BI-78D3 (30 and 60 mg/kg, i.p., twice daily) inhibited ear swelling by 45% and 70% respectively, compared to vehicle controls [1]
Immunohistochemical analysis of ear tissues showed that BI-78D3 (60 mg/kg) reduced phosphorylation of c-Jun and infiltration of inflammatory cells [1]

The LanthaScreen c-Jun (1-79) HeLa and LanthaScreen ATF2 (19-106) A549 cell lines, which stably express GFP-c-Jun 1-79 and GFP-ATF2 19-106, respectively, are used for the cell-based kinase assays for c-Jun and ATF2 phosphorylation. By measuring the time resolved FRET (TR-FRET) between a terbium labeled phospho-specific antibody and the GFP-fusion protein, phosphorylation is identified. 10,000 cells per well in 32 μl of assay medium (supplemented with 1% charcoal/dextran-treated FBS, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25 mM Hepes, pH 7.3, and devoid of phenol red) are plated on white tissue culture-treated 384-well plates. After overnight incubation, cells are pretreated for 60 minutes with BI-78D3 (0.001, 0.01, 0.1, 1, 10, and 100 μM), and then stimulated for 30 minutes with TNF-α, which activates both JNK and p38. After aspirating the medium out of the cells, 20 μL of lysis buffer (20 mM Tris•HCl, pH 7.6, 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) is added. Anti-pc-Jun (pSer73) or anti-pATF2 (pThr71) detection antibodies that have been terbium-labeled are present in the lysis buffer at a concentration of 2 nM. TR-FRET emission ratios are calculated on a BMG Pherastar fluorescence plate reader (excitation at 340 nm, emission at 520 and 490 nm; 100 s lag time, 200 s integration time, emission ratio=Em520/Em 490)[1] after allowing the assay to equilibrate for 1 h at room temperature.

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Fluorescence Polarization (FP) assay was used to measure JNK-JIP interaction inhibition. A fluorescently labeled JIP-derived peptide was incubated with recombinant JNK1/2/3 in assay buffer. Serial dilutions of BI-78D3 were added, and the mixture was incubated at room temperature for 1 hour. FP signals were measured to determine the ability of the compound to displace the peptide, and Ki values were calculated using binding isotherm models [1]
JNK kinase activity assay: Recombinant JNK1 was incubated with ATP, MgCl2, and GST-c-Jun substrate in the presence of BI-78D3 (0–100 μM). After 30 minutes at 37°C, the reaction was terminated, and phosphorylated GST-c-Jun was detected by Western blot to assess catalytic inhibition [1]

Mice
Male BL/6 mice aged 6 to 8 weeks are given a 10 mg/kg i.v. injection of ConA and BI-78D3. Mice are given isofluorane anesthesia before undergoing midventral laparotomy and having the left lateral and median lobes of the liver removed. Animals are put to death, their livers are surgically removed, and blood is obtained by puncturing their hearts. The concentration of alanine-aminotranferase in serum is determined[1].
Male, eleven weeks old OlaHsd db/db BKS.Cg-+Leprdb/+Leprdb Based on blood glucose levels adjusted three days prior to drug administration, mice are randomly assigned. A portable glucose meter is used to measure blood sugar levels. Mice are fasted for 6 hours prior to receiving 25 mg/kg of BI-78D3 intravenously (i.p.). Bovine Insulin (I-0516) is injected intravenously for 30 minutes following the administration of the test substance at a dose of 0.75 mg/kg. At predetermined intervals, blood samples are drawn, and blood glucose levels are measured as specified. Three hours after the test article is administered, food is returned[1].

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HeLa cell JNK signaling assay: HeLa cells were seeded in 6-well plates and allowed to adhere overnight. Cells were pretreated with BI-78D3 (0.1–50 μM) for 1 hour, then stimulated with anisomycin (1 μM) for 30 minutes. Cell lysates were prepared, and phosphorylated c-Jun (Ser63/73) and total c-Jun were detected by Western blot [1]
AP-1 luciferase reporter assay: HeLa cells were cotransfected with AP-1 luciferase reporter plasmid and Renilla luciferase control plasmid. After 24 hours, cells were pretreated with BI-78D3 for 1 hour, followed by anisomycin stimulation for 6 hours. Luciferase activity was measured using a dual-luciferase assay system to calculate IC50 [1]
A549 IL-8 production assay: A549 cells were seeded in 96-well plates and starved for 12 hours. BI-78D3 (0.5–50 μM) was added, and cells were stimulated with TNFα (10 ng/mL) for 24 hours. Supernatants were collected, and IL-8 levels were measured by ELISA [1]

25 mg/kg; I.V. injection
Mouse models of type 2 diabetes

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LPS-induced TNFα production model: Male C57BL/6 mice were randomized into vehicle and treatment groups. BI-78D3 was formulated in 10% DMSO + 90% corn oil and administered intraperitoneally at 50 mg/kg twice daily (12 hours apart). One hour after the second dose, mice were injected intraperitoneally with LPS (10 μg/mouse). Serum was collected 2 hours after LPS injection, and TNFα levels were measured by ELISA [1]
Mouse CHS model: Female BALB/c mice were sensitized with 2,4-dinitrofluorobenzene (DNFB) on the abdomen on day 0. On day 5, mice were challenged with DNFB on the ears. BI-78D3 (30, 60 mg/kg) or vehicle was administered intraperitoneally twice daily from day 4 to day 6. Ear thickness was measured 24 hours after challenge, and ear tissues were collected for immunohistochemistry [1]

In a 7-day in vivo study, BI-78D3 at doses up to 60 mg/kg (intraperitoneal injection, twice daily) did not cause significant changes in body weight or toxic clinical symptoms in mice [1].

[1]. Identification of a new JNK inhibitor targeting the JNK-JIP interaction site. Proc Natl Acad Sci U S A, 2008 Oct 28, 105(43):16809-13.

4-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-[(5-nitro-2-thiazolyl)thio]-1H-1,2,4-triazol-5-one is an aryl thioether. BI-78D3 is a novel allosteric inhibitor that targets the JNK-JIP interaction site, unlike ATP-competitive JNK inhibitors [1]. Its mechanism of action involves blocking the scaffold function of JNK, thereby inhibiting JNK-mediated downstream signaling without affecting the catalytic activity of JNK [1]. This compound shows potential for treating inflammatory diseases by inhibiting the production of pro-inflammatory cytokines and the infiltration of inflammatory cells [1].

C13H9N5O5S2

379.37

379.005

C, 41.16; H, 2.39; N, 18.46; O, 21.09; S, 16.90

883065-90-5

2747117

White to off-white solid powder

1.922g/cm3

705.721ºC at 760 mmHg

380.606ºC

1.879

2.37

1

9

3

25

588

0

O=C1N(C2C=C3C(OCCO3)=CC=2)C(SC2SC([N+](=O)[O-])=CN=2)=NN1

QFRLDZGQEZCCJZ-UHFFFAOYSA-N

InChI=1S/C13H9N5O5S2/c19-11-15-16-12(25-13-14-6-10(24-13)18(20)21)17(11)7-1-2-8-9(5-7)23-4-3-22-8/h1-2,5-6H,3-4H2,(H,15,19)

4-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-[(5-nitro-1,3-thiazol-2-yl)sulfanyl]-1H-1,2,4-triazol-5-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)