产品描述
CHIR-99021 is a GSK-3α/β inhibitor (IC50: 10/6.7 nM).

体外活性
CHIR 99021 inhibited human GSK-3β (Ki: 9.8 nmol/L). It exhibited from 500-fold to >10,000-fold selectivity for GSK-3 versus 20 other protein kinases [1]. CHIR99021 can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors, Oct4 and Klf4. When combined with Parnate, an inhibitor of lysine-specific demethylase 1, CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors, Oct4 and Klf4 [2]. In the presence of CHIR-99021 the viability of the ES-D3 cells was reduced by 24.7% at 2.5 μM, 56.3% at 5 μM, 61.9% at 7.5 μM and 69.2% at 10 μM CHIR-99021 with an IC50 of 4.9 μM. In ES-D3 cells cultivation with CHIR-99021 resulted in significant activation of the Wnt/beta-catenin pathway [3].

体内活性
In ZDF rats, a single oral dose of CHIR 99021 rapidly lowered plasma glucose, with a maximal reduction of nearly 150 mg/dl 3–4 h after administration. Importantly, reduced fasting hyperglycemia was achieved while plasma insulin remained at or below control levels throughout the time course of the experiment. The response was reproducible and dose-related (e.g., mild lowering at 8 mg/kg and maximal lowering at 30–48 mg/kg) [1].

激酶实验
Kinases were purified from SF9 cells through the use of their His or Glu tag. Glu-tagged proteins were purified as described, and His-tagged proteins were purified according to the manufacturer's instructions. 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 Me2SO, 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)/(Me2SO control ? no enzyme control) [4].

细胞实验
The Wnt/beta-catenin reporter assay was performed with the M50 Super 8× TOPFlash and M51 Super 8× FOPFlash vector containing the firefly luciferase gene under the control of TCF/LEF binding sites or mutated bindings sites. 12,500 cells were seeded overnight on gelatine-coated 96-well plates in LIF-containing ES cell medium. On the next day, the cells were transfected using Lipofectamine with one of the aforementioned vectors plus pGL4.75 [hRluc/CMV] encoding the renilla luciferase reporter gene hRluc as a transfection control. Six hours after transfection the medium was changed to medium devoid of LIF, with reduced serum, and supplemented with 5 μM CHIR-99021. The Dual-Luciferase? reporter assay system was employed 48 and 72 h after the medium change to follow the luminescence reaction using a GloMax?-multi detection system [4].
动物实验
Blood was obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose was measured directly or heparinized plasma was collected for measurement of glucose or insulin. Animals were pre-bled and randomized to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals fasted throughout the procedure with food removal early in the morning, 3 h before the first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). When the time course of plasma glucose and insulin changes in fasting ZDF rats was measured, food was removed ～16 h before test agent administration. The glucose challenges in the GTT were 1.35 g/kg i.p. (ipGTT) or 2 g/kg via oral gavage (oGTT). CHIR-99021 were formulated as solutions in 20 mmol/l citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose [1].

Cas No.
252917-06-9

分子式
C22H18Cl2N8

分子量
465.34

别名
CT99021;CHIR-99021

参考文献
[1]Bennett CN, et al. Regulation of Wnt signaling during adipogenesis. J Biol Chem. 2002 Aug 23;277(34):30998-1004.
[2]Ring DB, et al. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes. 2003 Mar;52(3):588-95.
[3]Gong-Bo Fu, Wei-Jian Huang, Min Zeng, Xu Zhou, Hong-Ping Wu, Chang-Cheng Liu, Han Wu, Jun Weng, Hong-Dan Zhang, Yong-Chao Cai, Charles Ashton, Min Ding, Dan Tang, Bao-Hua Zhang, Yi Gao, Wei-Feng Yu, Bo Zhai, Zhi-Ying He, Hong-Yang Wang, and He-Xin Yan . Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens [J]. Cell Research. 2019 Jan;29(1):8-22.
[4]Wei-jian L I, Zhen-yu W, Tian-jie Y, et al. The study of immortalized hepatocyte-derived liver progenitor-like cells used in bioartificial liver therapy[J]. Chinese Hepatolgy. 24(8): 871.
[5]Naujok O, et al. Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes. 2014 Apr 29;7:273.
[6]Li W, et al. Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2. Stem Cells. 2009 Dec;27(12):2992-3000.

引用文献
[1]Fu G B, Huang W J, Zeng M, et al. Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens. Cell Research. 2019, 29(1): 8-22
[2]Wu M, Zhang X, Zhang W, et al. Cancer Stem Cell Regulated Phenotypic Plasticity Protects Metastasized Cancer Cells from Ferroptosis. Nature Communications. 2022, 13(1): 1-16.
[3]Ma X, Lu Y, Zhou Z, Human expandable pancreatic progenitor–derived β cells ameliorate diabetes. Science Advances. 2022, 8(8): eabk1826.
[4]Yuan Y, Chen H, Ou S, et al. Generation of mitochondria-rich kidney organoids from expandable intermediate mesoderm progenitors reprogrammed from human urine cells under defined medium. Cell & Bioscience. 2022, 12(1): 1-20.
[5]He W, Zhu X, Xin A, et al. Long-term maintenance of human endometrial epithelial stem cells and their therapeutic effects on intrauterine adhesion. Cell & Bioscience. 2022, 12(1): 1-20.
[6]Wang W, Ren S, Lu Y, et al. Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production. The EMBO Journal. 2021 Jun 1;40(11):e106771. doi: 10.15252/embj.2020106771. Epub 2021 Apr 28.
[7]Lin R, Zhai Z, Kuang J, et al. H3K27ac mediated SS18/BAFs relocation regulates JUN induced pluripotent-somatic transition. Cell & Bioscience. 2022, 12(1): 1-14
[8]Yang Z, Liu H, Song R, et al. Reduced MAGI3 level by HPV18E6 contributes to Wnt/β‐catenin signaling activation and cervical cancer progression. FEBS Open bio. 2021, 11(11): 3051.

储存和溶解度
DMSO:9.3 mg/mL (20 mM)
Powder: -20°C for 3 years
In solvent: -80°C for 2 years

Note
For research use only .