GSK-3β (IC50 = 6.7 nM); GSK-3α (IC50 = 10 nM); cdc2 (IC50 = 8800 nM)

CHIR-99021 shows greater than 500-fold selectivity for GSK-3 versus its closest homologs CDC2 and ERK2, as well as other protein kinases. CHIR-99021 exhibits only modest inhibition of 23 nonkinase enzymes and only weak binding to a panel of 22 pharmacologically significant receptors. In CHO-IR cells that express the insulin receptor, CHIR-99021 causes the activation of glycogen synthase (GS) with an EC50 of 0.763 μM[1].

In a rodent model of type 2 diabetes, oral administration of CHIR-99021 at 30 mg/kg improves glucose metabolism. Three to four hours after oral administration, the maximum plasma glucose reduction—roughly 150 mg/dl—occurs, and plasma insulin levels stay at or below control. In ZDF rats, oral administration of CHIR-99021 at doses of 16 or 48 mg/kg an hour prior to oral glucose challenges significantly improves glucose tolerance, with plasma glucose levels falling by 14% and 33% at the 16 mg/kg and 48 mg/kg doses, respectively. The higher dose of CHIR-99021 also lessens hyperglycemia prior to the oral glucose challenge[1].

All kinase assays followed the same core protocol with variations in peptide substrate and activator concentrations described below. Polypropylene 96-well plates were filled with 300 μl/well buffer (50 mmol/l tris HCl, 10 mmol/l MgCl2, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 25 mmol/l β-glycerophosphate, 1 mmol/l NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. Information on the kinase concentration, peptide substrate, and activator (if applicable) for these assays is as follows: GSK-3α (27 nmol/l, and 0.5 μmol/l biotin-CREB peptide); GSK-3β (29 nmol/l, and 0.5 μmol/l biotin-CREB peptide); cdc2 (0.8 nmol/l, and 0.5 μmol/l biotin histone H1 peptide); erk2 (400 units/ml, and myelin basic protein-coated Flash Plate [Perkin-Elmer]); PKC-α (1.6 nmol/l, 0.5 μmol/l biotin-histone H1 peptide, and 0.1 mg/ml phosphatidylserine + 0.01 mg/ml diglycerides); PKC-ζ (0.1 nmol/l, 0.5 μmol/l biotin-PKC-86 peptide, and 50 μg/ml phosphatidylserine + 5 μg/ml diacylglycerol); akt1 (5.55 nmol/l, and 0.5 μmol/l biotin phospho-AKT peptide); p70 S6 kinase (1.5 nmol/l, and 0.5 μmol/l biotin-GGGKRRRLASLRA); p90 RSK2 (0.049 units/ml, and 0.5 μmol/l biotin-GGGKRRRLASLRA); c-src (4.1 units/ml, and 0.5 μmol/l biotin-KVEKIGEGTYGVVYK); Tie2 (1 μg/ml, and 200 nmol/l biotin-GGGGAPEDLYKDFLT); flt1 (1.8 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); KDR (0.95 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); bFGF receptor tyrosine kinase (RTK; 2 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); IGF1 RTK (1.91 nmol/l, and 1 μmol/l biotin-GGGGKKKSPGEYVNIEFG-amide); insulin RTK (using DG44 IR cells); AMP kinase (470 units/ml, 50 μmol/l SAMS peptide, and 300 μmol/l AMP); pdk1 (0.25 nmol/l, 2.9 nmol/l unactivated Akt, and 20 μmol/l each of DOPC and DOPS + 2 μmol/l PIP3); CHK1 (1.4 nmol/l, and 0.5 μmol/l biotin-cdc25 peptide); CK1-ε (3 nmol/l, and 0.2 μmol/l biotin-peptide); DNA PK; and phosphatidylinositol (PI) 3-kinase (5 nmol/l, and 2 μg/ml PI). Test compounds or controls were added in 3.5 μl of DMSO, followed by 50 μl of ATP stock to yield a final concentration of 1 μmol/l ATP in all cell-free assays. After incubation, triplicate 100-μl aliquots were transferred to Combiplate eight plates (LabSystems, Helsinki, Finland) containing 100 μl/well 50 μmol/l ATP and 20 mmol/l EDTA. After 1 h, the wells were rinsed five times with PBS, filled with 200 μl of scintillation fluid, sealed, left 30 min, and counted in a scintillation counter. All steps were performed at room temperature. Inhibition was calculated as 100% × (inhibited − no enzyme control)/(DMSO control − no enzyme control).[1]

CHO-IR cells expressing human insulin receptor are grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. In 2-ml of medium devoid of fetal bovine serum, trypsinized cells are seeded in 6-well plates at a density of 1×106 cells per well. Following 24 hours, the medium is changed to 1 ml of serum-free medium containing the GSK-3 inhibitor or a control (final DMSO concentration <0.1%) for 30 min at 37°C. Lysing and centrifuging the cells for 15 min. at 4 °C/14000g. Using the filter paper assay developed by Thomas et al., the activity ratio of GS is calculated as the difference between the activity of GS in the presence and absence of 5 mmol/l glucose-6-phosphate.

Female db/db mice; Male ZDF rats
8-48 mg/kg
Oral administration [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[2].

[1]. Selective glycogen synthase kinase 3 inhibitors potentiate activation of glucose transport and utilization in vitro and in vivo. Diabetes. 2003 Mar;52(3):588-95.

[2]. 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.

[3]. Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains. PLoS One. 2012;7(4):e35892.

[4]. Regulation of Wnt signaling during adipogenesis. J Biol Chem. 2002 Aug 23;277(34):30998-1004.

[5]. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation. Sci Rep. 2015 Apr 10;5:8566.

CHIR 99021 is a member of the class of aminopyrimidines that is 2-aminopyrimidine substituted at positions N2, 5 and 6 by (5-cyanopyridin-2-yl)ethyl, 4-methylimidazol-2-yl and 2,4-dichlorophenyl groups respectively. It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor. It is a member of imidazoles, a dichlorobenzene, an aminopyrimidine, an aminopyridine, a cyanopyridine, a secondary amino compound and a diamine.

---

Background: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/principal findings: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/significance: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.[3]

C22H18CL2N8

465.34

464.103

C, 56.78; H, 3.90; Cl, 15.24; N, 24.08

252917-06-9

252917-06-9;1782235-14-6 (3HCl);2109414-84-6 (2HCl);1797989-42-4 (HCl);

9956119

Off-white to light brown solid powder

1.5±0.1 g/cm3

784.1±70.0 °C at 760 mmHg

428.0±35.7 °C

0.0±2.7 mmHg at 25°C

1.700

3.98

3

7

7

32

645

0

ClC1C([H])=C(C([H])=C([H])C=1C1C(=C([H])N=C(N=1)N([H])C([H])([H])C([H])([H])N([H])C1C([H])=C([H])C(C#N)=C([H])N=1)C1=NC([H])=C(C([H])([H])[H])N1[H])Cl

AQGNHMOJWBZFQQ-UHFFFAOYSA-N

InChI=1S/C22H18Cl2N8/c1-13-10-29-21(31-13)17-12-30-22(32-20(17)16-4-3-15(23)8-18(16)24)27-7-6-26-19-5-2-14(9-25)11-28-19/h2-5,8,10-12H,6-7H2,1H3,(H,26,28)(H,29,31)(H,27,30,32)

6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

View More

Solubility in Formulation 3: ~3.5 mg/mL (7 mM) in 4%DMSO+30%PEG 300+ddH2O, clear solution
Solubility in Formulation 4: ~5 mg/mL (10.7 mM) in 0.5% CMC-Na/saline water, suspension solution
Solubility in Formulation 5: ≥ 2.1 mg/mL (4.5 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline, clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21 mg/mL of DMSO stock solution and add tO + 400 μL of PEG300, mix well (clear solution); Then add 50 μL of Tween 80 to the above solution, mix well (clear solution); Finally, add 450 μL of saline to the above solution, mix well (clear solution).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Solubility in Formulation 6: ≥ 2.1 mg/mL (4.5 mM) in 10% DMSO + 90% Corn oil, clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21 mg/mL of DMSO stock solution and add to 900 μL of corn oil, mix well (clear solution).

---

Solubility in Formulation 7: 5 mg/mL (10.74 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 8: 5 mg/mL (10.74 mM) in 20% SBE-β-CD adjusted to pH 4-4.5 with 1 N acetic (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)