CHIR-99021 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.

IC50: 10 nM/6.7 nM (GSK-3α/β)^[1]

CHIR 99021inhibits human GSK-3β with K_i 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].

• [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.

Kinases are purified from SF9 cells through use of their His or Glu tag. Glu-tagged proteins are purified, and His-tagged proteins are purified. Kinase assays are 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 mMdithiothreitol, 25 mMβ-glycerophosphate, 1 mM NaF, and 0.01% bovine serum albumin). Peptides has K_m values from 1 to 100 μM. CHIR 99021 or CHIR GSKIA is added in 3.5 μL of Me₂SO, followed by ATP to a final concentration of 1 μM. After incubation, triplicate 100-μL aliquots are transferred to Combiplate 8 plates containing 100 μL/well of 50 μM ATP and 20 mM EDTA. After 1 hour, the wells are 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 are at room temperature. The percentage of inhibition is calculated as 100×(inhibitor-no enzyme control)/(Me₂SO control-no enzyme control)^[2]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

CHIR 99021 is dissolved in DMSO and stored, and then diluted with appropriate media before use^[3].

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]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

CHIR 99021 is formulated as solutions in 20 mM citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose (Rat)^[1].
CHIR 99021 is prepared in DMSO and diluted (Mice)^[4].

Rat^[1]
Primary hepatocytes from male Sprague Dawley rats that weighed <140 g="" are="" prepared="" and="" used="" 1-3="" h="" after="" isolation.="" aliquots="" of="">⁶cells in 1 mL of DMEM/F12 medium plus 0.2% BSA and CHIR 99021(orally at 16 or 48 mg/kg) or controls are incubated in 12-well plates on a low-speed shaker for 30 min at 37°C in a CO₂-enriched atmosphere, collected by centrifugation and lysed by freeze/thaw in buffer A plus 0.01% NP40; the GS assay is again performed.
Mice^[4]
Mice 6-10 weeks old are used. The 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. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [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.

465.34

C₂₂H₁₈Cl₂N₈

252917-06-9

Room temperature in continental US; may vary elsewhere

DMSO: ≥ 5.1 mg/mL

* "<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">