CHIR-98014 (CT98014) is a selective GSK3 inhibitor; potentiate insulin activation of glucose transport and utilization in vitro and in vivo.

GSK-3α:0.65 nM, GSK-3β:0.58 nM

CHIR 98014降低高血糖和改善葡萄糖利用的效果并不限于db/db小鼠和ZDF大鼠,由于观察到ob/ob小鼠,饮食诱导的糖尿病C57BL/6小鼠和用CHIR-2处理的葡萄糖不耐受的SHHF大鼠出现类似结果.此外,CHIR-98014降低了出生后大鼠皮质和海马中tau蛋白的磷酸化（Ser396）.GSK-3抑制剂CHIR-98014作用于从瘦弱的患有糖尿病和抗胰岛素的ZDF鼠分离的I型骨骼肌,激活GS活性.瘦弱患有糖尿病鼠分离的肌肉中,胰岛素和CHIR 98014激活的GS比ZDF鼠分离的肌肉中强很多.CHIR 98014或胰岛素作用于这些细胞和肌肉不会改变全部GS活性. 同时,CHIR 98014不会影响从瘦弱动物分离的肌肉中胰岛素剂量反应.

虽然CHIR-98014作为ATP结合的简单竞争性抑制剂,但它对GSK-3显示出相对于20种其他蛋白激酶（包括Cdc2,ERK2,Tie-2和KDR在内）500倍至> 1000倍的选择性。CHIR 98014抑制人类GSK-3β,Ki值为0.87 nM。CHIR 98014抑制CDC2,IC50为3.7 μM。然而,CHIR 98014对GSK-3高度同源的ɑ和β亚型具有相似效果,但是在GSK-3和最接近的同系物cdc2和erk2间具有明显区别。CHIR 98014使CHO-IR和鼠肝细胞达到半数最大GS刺激反应的浓度分别为106和107 nM。将表达胰岛素受体的CHO-IR细胞或原代大鼠肝细胞暴露于递增浓度的抑制剂CHIR98014中导致GS活性比率高于基础值的两倍至三倍的刺激。

Kinase assays: Polypropylene 96-well plates are ?lled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. CHIR-98014 or controls are added in 3.5 μL of DMSO, followed by 50 μL of ATP stock to yield a ?nal concentration of 1 μM ATP in all cell-free assays. After incubation, triplicate 100-μL aliquots are transferred to Combiplate eight plates containing 100 μL/well 50 μM ATP and 20 mM EDTA. After 1 hour, the wells are rinsed ?ve times with PBS, ?lled with 200 μL of scintillation ?uid, sealed, left 30 min, and counted in a scintillation counter. All steps are performed at room temperature.

CHO-IR cells expressing human insulin receptor are grown to 80% con?uence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. Trypsinized cells are seeded in 6-well plates at 1 × 106 cells/well in 2 mL of medium without fetal bovine serum. After 24 hours, medium is replaced with 1 mL of serum-free medium containing GSK-3 inhibitor CHIR 98014 or control (?nal DMSO concentration 0.1%) for 30 min at 37 °C. Cells are lysed by freeze/thaw in 50 mM tris (pH 7.8) containing 1 mM EDTA, 1 mM DTT, 100 mM NaF, 1 mM phenylmethylsulfonyl ?uoride, and 25 μg/mL leupeptin (buffer A) and centrifuged 15 min at 4 °C/14000 g. The activity ratio of GS is calculated as the GS activity in the absence of glucose-6-phosphate divided by the activity in the presence of 5 mM glucose-6-phosphate. (Only for Reference)