CHIR-99021 HCl

Biological Activity

CHIR-99021 exhibits >500-fold selectivity for GSK-3 over closely related kinases. CHIR-99021 also displays >800-fold selectivity against 45 additional enzymes and receptors. CHIR-99021 (3 μM) increases free cytosolic β-catenin by 1.9-fold, mimicking the canonical Wnt signaling pathway in 3T3-L1 preadipocytes. CHIR-99021 promotes primary beta cell replication in isolated rat islets at concentrations as low as 1 μM, with 2-3 fold increase of cell replication at 5 μM of CHIR-99021 treatment. Oral administration of CHIR-99021 at 16 or 48 mg/kg 1 hour before oral glucose challenges in ZDF rats significantly improves glucose tolerance with 14% and 33% reduction in plasma glucose at 16 mg/kg and 48 mg/kg, respectively, and the higher dose of CHIR-99021 also reduces hyperglycemia before the oral glucose challenge.

Product Citations

• 

  Nature. 2022 Jul;607(7917):149-155.

  Deciphering the immunopeptidome in vivo reveals new tumour antigens
  CHIR-99021 HCl purchased from AbMole

• 

  Nat Commun. 2022 Oct 20;13(1):6230.

  Acute deletion of TET enzymes results in aneuploidy in mouse embryonic stem cells through decreased expression of Khdc3
  CHIR-99021 HCl purchased from AbMole

• 

  Mol Biol Cell. 2022 Jul 1;33(8):mbcP22031008.

  Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models
  CHIR-99021 HCl purchased from AbMole

• 

  2020 Sep.

  Developing a model to assess the contribution of cytokeratin 19-expressing cells during multipotent stromal cell-induced islet regeneration
  CHIR-99021 HCl purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Sci Rep (2015). Figure 3. CHIR-99021
	Method	qPCR
	Cell Lines	J1 mESCs and F9 mEC cells
	Concentrations	3 μM
	Incubation Time	24 h
	Results	By contrast, all of the ESCC miRNAs were downregulated by CHIR (Fig. 3a–e). The miR-200b-429 and miR-183-182 clusters, which are highly expressed in pluripotent cells, were downregulated by both BIO and CHIR (Fig. 3f, 3g).

	Source	Sci Rep (2015). Figure 1. CHIR-99021
	Method	immunofluorescence staining
	Cell Lines	J1 mESCs and F9 mEC cells
	Concentrations	3 μM
	Incubation Time	24 h
	Results	The relativemRNA expression level of Nanog was determined in response to BIO or CHIR in J1mESCs and F9mEC cells by qPCR. As expected, transcription of Nanog in J1 mESCs and F9 mEC cells was upregulated by both BIO and CHIR

Protocol (for reference only)

Cell Experiment
Cell lines	CHO-IR cells
Preparation method	GS activity assays. CHO-IR cells expressing human insulin receptor, (provided by Hans Bos) were grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine (34). Trypsinized cells were seeded in 6-well plates at 1 × 106 cells/well in 2 ml of medium without fetal bovine serum. After 24 h, medium was replaced with 1 ml of serum-free medium containing GSK-3 inhibitor or control (final DMSO concentration <0.1%) for 30 min at 37°C. Cells were lysed by freeze/thaw in 50 mmol/l tris (pH 7.8) containing 1 mmol/l EDTA, 1 mmol/l DTT, 100 mmol/l NaF, 1 mmol/l phenylmethylsulfonyl fluoride, and 25 μg/ml leupeptin (buffer A) and centrifuged 15 min at 4°C/14000g. The activity ratio of GS was calculated as the GS activity in the absence of glucose-6-phosphate divided by the activity in the presence of 5 mmol/l glucose-6-phosphate, using the filter paper assay of Thomas et al.
Concentrations	0~500nM
Incubation time	30min

Animal Experiment
Animal models	fasting ZDF rats
Formulation	20 mmol/l citrate-buffered 15% Captisol or suspensions in 0.5% carboxymethylcellulose
Dosages	30 mg/kg
Administration	oral gavage

Chemical Information

Molecular Weight	501.8
Formula	C22H18Cl2N8.HCl
CAS Number	1797989-42-4
Solubility (25°C)	DMSO 60 mg/mL Water 6 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Ring DB, et al. Diabetes. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo.