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]. Diabetes. 2003 Mar;52(3):588-95.

C22H18CL2N8

465.34

464.10315

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);

Off-white to light brown solid powder

4.3

115Ų

CC1=CN=C(N1)C2=CN=C(N=C2C3=C(C=C(C=C3)Cl)Cl)NCCNC4=NC=C(C=C4)C#N

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: Formulation 1: ~3.5 mg/mL (7 mM) in 4%DMSO+30%PEG 300+ddH2O, clear solution
Formulation 2: ~5 mg/mL (10.7 mM) in 0.5% CMC-Na/saline water, suspension solution
Formulation 3: ≥ 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.
Formulation 4: ≥ 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 4: 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 5: 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.)