CHIR-98014

• Catalog No.: S2745
• CAS Number: 252935-94-7
• M. F.: C20H17Cl2N9O2
• M. W.: 486.31408
• Storage: -20°C

SYNONYMS

• IUPAC name: 2-N-(2-{[4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-yl]amino}ethyl)-5-nitropyridine-2,6-diamine
• IUPAC Traditional name: 2-N-(2-{[4-(2,4-dichlorophenyl)-5-(imidazol-1-yl)pyrimidin-2-yl]amino}ethyl)-5-nitropyridine-2,6-diamine

DATABASE IDS

• CAS Number: 252935-94-7

PROPERTIES

• Target: GSK-3
• Salt Data: Free Base
• Storage Condition: -20°C

DETAILS

Description (English)

Research Area

• Description: Cancer

Biological Activity

• Description: CHIR-98014 is a potent and selective GSK-3 inhibitor for GSK-3ɑ and GSK-3β with IC50 of 0.65 nM and 0.58 nM, respectively.
• Targets:

  GSK-3ɑ (Human)

  ;

  GSK-3β (Human)

• IC50:

  0.65 nM

  ;

  0.58 nM ^[1]

• In Vitro: CHIR-98014 inhibits human GSK-3β with K_i of 0.87 nM. CHIR-98014 is very effective in preventing murine and rat GSK-3. Although CHIR-98014 acts as a simple competitive inhibitor of ATP binding, it displays from 500-fold to >1000-fold selectivity for GSK-3 versus 20 other protein kinases including Cdc2, ERK2, Tie-2 and KDR. CHIR-98014 prevents Cdc2 with IC50 of 3.7 μM. However, CHIR 98014 reveals similar ptoency against the highly homologous ɑ and β isoforms of GSK-3, it is noteworthy that it stronly discriminated between GSK-3 and its closest homologs CDC2 and ERK2. Exposure of insulin receptor-expressing CHO-IR cells or primary rat hepatocytes to increasing concentrations of inhibitor CHIR98014 results in a two- to three-fold stimulation of the GS activity ratio above basal. The concentrations of CHIR-98014 giving rise to half-maximal GS stimulation (EC50) is 106 nM and 107 nM for CHO-IR and rat hepatocytes, respectively. ^[1]
• In Vivo: GSK-3 inhibitor CHIR-98014 activates the GS activity ratio in isolated type I skeletal muscle from insulin-sensitive lean Zucker and from insulin-ressitant ZDF rats. Soleus muscle isolated from ZDF rats shows significant resistance to insulin for activation of GS but responded to 500 nM CHIR-98014 to the same extent (40% increase) as muscle from lean Zucker rats. Notably, GS activation by insulin plus CHIR-98014 is additive in muscle from lean Zucker rats and greater than additive in muscle from the ZDF rats. Total GS activity is not altered by either CHIR-98014 or insulin in these cells and muscles. Meanwhile, CHIR-98014 does not influence the insulin dose-response in muscle from lean animals. The reduction in hyperglycemia and improved glucose disposal are not limited to db/db mice and ZDF rats, as similar results are observed with ob/ob mice, diet-induced diabetic C57BL/6 mice, and glucose-intolerant SHHF rats treated with CHIR-98014. ^[1] Additionally, CHIR-98014 decreases the phosphorylation (Ser396) of tau protein in the cortex and hippocampus of postnatal rats. ^[2]

Protocol

• Protocol: Kinase Assay ^[1]
• Kinase assays: Polypropylene 96-well plates are ?lled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. CHIR-98014 or controls are added in 3.5 μL of DMSO, followed by 50 μL of ATP stock to yield a ?nal concentration of 1 μM ATP in all cell-free assays. After incubation, triplicate 100-μL aliquots are transferred to Combiplate eight plates containing 100 μL/well 50 μM ATP and 20 mM EDTA. After 1 hour, the wells are rinsed ?ve times with PBS, ?lled with 200 μL of scintillation ?uid, sealed, left 30 min, and counted in a scintillation counter. All steps are performed at room temperature.
• Protocol: Cell Assay ^[1]
• Cell Lines: CHO-IR cells
• Concentrations: 0.01-10 μM
• Incubation Time: 30 min
• Methods:

  CHO-IR cells expressing human insulin receptor are grown to 80% con?uence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. Trypsinized cells are seeded in 6-well plates at 1 × 10^6 cells/well in 2 mL of medium without fetal bovine serum. After 24 hours, medium is replaced with 1 mL of serum-free medium containing GSK-3 inhibitor CHIR 98014 or control (?nal DMSO concentration 0.1%) for 30 min at 37 °C. Cells are lysed by freeze/thaw in 50 mM tris (pH 7.8) containing 1 mM EDTA, 1 mM DTT, 100 mM NaF, 1 mM phenylmethylsulfonyl ?uoride, and 25 μg/mL leupeptin (buffer A) and centrifuged 15 min at 4 °C/14000 g. The activity ratio of GS is calculated as the GS activity in the absence of glucose-6-phosphate divided by the activity in the presence of 5 mM glucose-6-phosphate.

• Protocol: Animal Study ^[1]
• Animal Models: db/db mice with 8-9 weeks
• Formulation: 15% Captisol/citrate vehicle
• Doses: 30 mg/kg
• Administration: Subcutaneously

References

• References: [1] Ring DB,et al. Diabetes. 2003, 52(3), 588-595.
• References: [2] Selenica ML, et al. Br J Pharmacol. 2007, 152(6), 959-979.

REFERENCES

• Ring DB,et al. Diabetes. 2003, 52(3), 588-595.
• Selenica ML, et al. Br J Pharmacol. 2007, 152(6), 959-979.