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1782235-14-6

1782235-14-6 structure
1782235-14-6 structure
  • Name: CHIR 99021 trihydrochloride
  • Chemical Name: 6-[(2-{[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino}ethyl)amino]nicotinonitrile trihydrochloride
  • CAS Number: 1782235-14-6
  • Molecular Formula: C22H21Cl5N8
  • Molecular Weight: 574.721
  • Catalog: Signaling Pathways Autophagy Autophagy
  • Create Date: 2018-07-06 19:54:34
  • Modify Date: 2024-01-08 13:17:31
  • CHIR-99021 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.

Name 6-[(2-{[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino}ethyl)amino]nicotinonitrile trihydrochloride
Synonyms 6-[(2-{[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino}ethyl)amino]nicotinonitrile trihydrochloride
3-Pyridinecarbonitrile, 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-, hydrochloride (1:3)
CHIR-99021 (trihydrochloride)
Description CHIR-99021 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.
Related Catalog
Target

GSK-3β:6.7 nM (IC50)

GSK-3α:10 nM (IC50)

cdc2:8800 nM (IC50)

In Vitro CHIR 99021inhibits 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 Vivo 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].
Kinase Assay 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 Km 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].
Cell Assay 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].
Animal Admin Rats[1] Primary hepatocytes from male Sprague Dawley rats that weighed <140 g are prepared and used 1-3 h after isolation. Aliquots of 1×106cells 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.
References

[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.

Molecular Formula C22H21Cl5N8
Molecular Weight 574.721
Exact Mass 572.033203
Storage condition 2-8℃