A selective GSK3 inhibitor
CHIR-99021 trihydrochloride (CT99021 trihydrochloride) is a GSK-3α/β inhibitor with IC50 of 10 nM/6.7 nM; >500-fold selectivity for GSK-3 versus its closest homologs CDC2 and ERK2, as well as other protein kinases.
CHIR-99021 inhibits human GSK-3β with Ki values of 9.8 nM[1]. CHIR-99021 is a small organic molecule that inhibits GSK3α and GSK3β by competing for their ATP-binding sites.In vitro kinase assays reveal that CHIR-99021 specifically inhibits GSK3β (IC50=~5 nM) and GSK3α (IC50=~10 nM), with little effect on other kinases[2]. In the presence of CHIR-99021 the viability of the ES-D3 cells is 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[3].
In ZDF rats, a single oral dose of CHIR-99021 (16 mg/kg or 48 mg/kg) rapidly lowers plasma glucose, with a maximal reduction of nearly 150 mg/dl 3-4 h after administration[1]. CHIR99021 (2 mg/kg) given once, 4 h before irradiation, significantly improves survival after 14.5 Gy abdominal irradiation (ABI). CHIR99021 treatment significantly blocks crypt apoptosis and accumulation of p-H2AX+ cells, and improves crypt regeneration and villus height. CHIR99021 treatment increases Lgr5+ cell survival by blocking apoptosis, and effectively prevents the reduction of Olfm4, Lgr5 and CD44 as early as 4 h[4].
参考文献:
[1]. 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.
[2]. Bennett CN, et al. Regulation of Wnt signaling during adipogenesis. J Biol Chem. 2002 Aug 23;277(34):30998-1004.
[3]. 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.
[4]. Wang X, et al. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation. Sci Rep. 2015 Apr 10;5:8566.
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Cell experiment: |
The viability of the mouse ES cells is determined after exposure to different concentrations of GSK3 inhibitors for three days using the MTT assay. The decrease of MTT activity is a reliable metabolism-based test for quantifying cell viability; this decrease correlates with the loss of cell viability. 2,000 cells are seeded overnight on gelatine-coated 96-well plates in LIF-containing ES cell medium. On the next day the medium is changed to medium devoid of LIF and with reduced serum and supplemented with 0.1-1 μM BIO, or 1-10 μM SB-216763, CHIR-99021 or CHIR-98014. Basal medium without GSK3 inhibitors or DMSO is used as control. All tested conditions are analyzed in triplicates[3]. |
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Animal experiment: |
Rats[1] Primary hepatocytes from male Sprague Dawley rats that weighed PUMA+/+ and PUMA-/- littermates on C57BL/6 background (F10) and Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice are subjected to whole body irradiation (TBI), or abdominal irradiation (ABI). Mice are injected intraperitoneally (i.p.) with 2 mg/kg of CHIR99021 4 h before radiation or 1 mg/kg of SB415286 28 h and 4 h before radiation. Mice are sacrificed to collect small intestines for histology analysis and western blotting. All mice are injected i.p. with 100 mg/kg of BrdU before sacrifice. |
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参考文献: [1]. 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. |
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