Description:
IC50: 150, 220, and 70 nM for hJNK1, hJNK2, and hJNK3, respectively
Recent evidence suggests activation of the c-Jun NH2-terminal protein kinase (JNK) signal transduction pathway may play a role in ischemia-induced cell death. Therefore, preventing the activation of JNK, or c-Jun phosphorylation could be neuroprotective. AS601245 is a c-Jun NH2-terminal protein kinase inhibitor.
In vitro: AS601245 demonstrated a nonspecific inhibition of the three JNK human isoforms. AS601245 inhibits isolated hJNK3 in an ATPcompetitive manner. Selectivity of AS601245 was tested against a large panel of kinases. It exhibited 10- to 20-fold selectivity over c-src, CDK2, and c-Raf and more than 50- to 100-fold selectivity over a range of Tyr- and Ser/Thr-protein kinases [1].
In vivo: AS601245 administered i.p. provided significant protection against the delayed loss of hippocampal CA1 neurons in a gerbil model of transient global ischemia. This effect is mediated by JNK inhibition and thus by c-Jun expression and phosphorylation. A significant neuroprotective effect of AS601245 administered either by i.p. injection or as i.v. bolus followed by an i.v. infusion was also observed in rats after focal cerebral ischemia [1].
Clinical trial: Up to now, AS601245 is still in the preclinical development stage.
Reference:
[1] Carboni S, Hiver A, Szyndralewiez C, Gaillard P, Gotteland JP, Vitte PA.? AS601245 (1,3-benzothiazol-2-yl (2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile): a c-Jun NH2-terminal protein kinase inhibitor with neuroprotective properties. J Pharmacol Exp Ther. 2004 Jul;310(1):25-32. Epub 2004 Feb 26.
Kinase experiment: | Rat JNK3 assays are performed in 96-well low binding Corning MTT plates: 0.5 μg of recombinant, preactivated GST-JNK3 is incubated with 1 μg of recombinant, biotinylated GST-c-Jun and 2 μM [33Pγ]ATP (2 nCi/μl), in the presence or absence of compounds according to formula I and in a reaction volume of 50 μL containing 50 mM Tris-HCl, pH 8.0; 10 mM MgCl2, 1 mM Dithiothreitol, and 100 μM NaVO4, for 120 min and at room temperature. The reaction is stopped by the addition of 200 μL of a solution containing 250 μg of Streptavidin-coated SPA beads, 5 mM EDTA, 0.1% Triton X-100, and 50 μM ATP, in phosphate saline buffer and further incubation at room temperature for 60 min. After incubation, beads are sedimented by centrifugation at 1500g for 5 min, resuspended in 200 μL of phosphate-buffered saline (PBS) containing 5 mM EDTA, 0.1% Triton X-100, and 50 μM ATP and the radioactivity is measured in a scintillation beta counter, following further sedimentation of the beads by settling down for 60 min at room temperature. Similar method is used to demonstrate inhibition of JNK1 and JNK2[1]. |
Cell experiment: | CaCo-2 cell proliferation is evaluated by using the kit “CellTiter-Glo Luminescent Cell Viability Assay”. This highly sensitive assay detects the luminescence released by the metabolically active cells. Quantification of luminescence is expressed as RLU (relative light unit). For the proliferation experiments, treatments are performed by adding the drugs (e.g., 0.1?μM AS601245) to the CaCo-2 cells seeded at about 4,000 cells/well in a 96-well plate. HepG2 cell proliferation is analysed through the MTT method. Briefly, 1500 cells/well are seeded in 200?μL of serum-supplemented media and the following day treated with the drugs (e.g, 0.1?μM AS601245). 20?μL of 5?mg/mL thiazolyl blue tetrazolium bromide is subsequently added to the cells and removed 2 hours later. 100?μL of DMSO is added to the cells, and the absorbance is recorded at 570?nm through a 96 well plate ELISA reader. Viability is evaluated through Trypan blue exclusion test[2]. |
Animal experiment: | Mice[1]C3H/HEN mice receive an oral treatment with AS601245 (0.3, 1, 3, or 10 mg/kg). Fifteen minutes later, Endotoxins (0.3 mg/kg) are i.p. injected. Heparinized whole blood is collected by retro orbital puncture under isoflurane anesthesia. TNF-α is determined in plasma using an enzyme-linked immunosorbent assay kit. Control animals receive 0.5% CMC/0.25% Tween 20 (10 mL/kg) as vehicle[1].Rats[1]Wistar rats are randomly divided into a vehicle-treated control group, a reference compound-treated group, and a drug-treated group. The reference compound-treated group receive MK-801 (3 mg/kg i.p.) administered 1 h postischemia onset. The drug-treated group receive AS601245 (6, 18, or 60 mg/kg) administered at the initiation of reperfusion and 5 h later. Control animals receive 0.9% saline (10 ml/kg i.p.).Wistar rats are randomly divided into three groups. The reference compound-treated group receive MK-801 (3 mg/kg i.p.) administered 1 h postischemia onset. The drug-treated group receive an intravenous bolus of AS601245 (1 mg/kg) injected at the initiation of reperfusion followed by an intravenous infusion with a flow of 0.6 mg/kg/h during 22 h. Control animals receive a bolus plus an intravenous infusion of 0.9% saline (10 mL/kg)[1]. |
参考文献: [1]. Carboni S, et al. AS601245 (1,3-benzothiazol-2-yl (2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile): a c-Jun NH2-terminal protein kinase inhibitor with neuroprotective properties. J Pharmacol Exp Ther. 2004 Jul;310(1):25-32. | |
动态评分
0.0