CHIR-99021 (GSK-3抑制剂) (RPM0009)

体外活性/In vitro:
1.用CHIR-99021（1-10 µM）处理小鼠干细胞ES-D3 72 h，MTT法检测细胞生长抑制情况。CHIR-99021剂量依赖性地抑制细胞生长，IC50为4.9 µM。[1]
2.用CHIR-99021（3 µM）处理小鼠胚胎干细胞J1 mESCs和小鼠胚胎瘤细胞F9 mEC 24 h，免疫荧光法检测靶点蛋白表达水平。CHIR-99021处理后，J1-mESCs和F9-mEC细胞的细胞质和细胞核中的β-连环蛋白增加。[2]
3.用CHIR-99021（5 µM）处理人Tenon成纤维细胞HTFs 48 h，Western Blot检测靶点蛋白表达水平。CHIR-99021处理使活性形式的GSK-3β（p-GSK-3β (Y216)）的产生显著减少。[3]

体内活性/In vivo:
1.将CHIR-99021（37.5 mg/kg/第0-3、6-10、13-17和20天每天两次）口服给药和paclitaxel（10 mg/kg/第0天单次给药）腹腔注射给携带人非小细胞肺癌肿瘤H1975的Balb/c nude小鼠，检测体内抗肿瘤活性。CHIR-99021和paclitaxel在体内协同作用，抑制NSCLC肿瘤的生长。[4]
2.将CHIR-99021（1-10 mg/kg）单次腹腔注射给有酒精或蔗糖自给药史的C57BL/6J小鼠，研究GSK-3的直接药理学抑制是否会改变酒精在小鼠体内的积极增强作用。CHIR-99021剂量依赖性地增加了酒精强化反应，而对蔗糖自我给药或运动活性没有影响，显著降低了pGSK-3β在所有测试脑区的表达，仅在NAcb中降低了PICK1并增加了GluA2的总表达。[5]

激酶实验/Kinase Assay:
Kinases were purified from SF9 cells through the use of their His or Glu tag. Kinase assays were performed in 96-well plates with appropriate peptide substrates in a 300 μL reaction buffer (variations on 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 25 mMβ-glycerophosphate, 1 mM NaF, and 0.01% bovine serum albumin). Peptides had Km values from 1 to 100 μM. CHIR 99021 or CHIR GSKIA was added in 3.5 μL of DMSO, followed by ATP to a final concentration of 1 μM. After incubation, triplicate 100 μL aliquots were transferred to Combiplate 8 plates containing 100 μL/well of 50 μM ATP and 20 mM EDTA. After 1 hour, the wells were rinsed five times with phosphate-buffered saline, filled with 200 μL of scintillation fluid, sealed, and counted in a scintillation counter 30 min later. All of the steps were at room temperature. The percentage of inhibition was calculated as 100 × (inhibitor-no enzyme control)/(DMSO control-no enzyme control) [4].

GSK-3α: 10 nM, GSK-3β: 6.7 nM