GSK-3β (IC50 = 0.58 nM); GSK-3α (IC50 = 0.65 nM); cdc2 (IC50 = 3700 nM)

CHIR-98014 inhibits human GSK-3β with Ki of 0.87 nM. CHIR-98014 is very effective in preventing murine and rat GSK-3. CHIR-98014 exhibits from a 500-fold to >1000-fold selectivity for GSK-3 versus 20 other protein kinases, such as Cdc2, ERK2, Tie-2, and KDR, despite acting as a straightforward competitive inhibitor of ATP binding. CHIR-98014 inhibits Cdc2 with an IC50 of 3.7 M. It is noteworthy that CHIR 98014 only distinguished between GSK-3 and its closest homologs CDC2 and ERK2, even though it exhibits similar potency against the highly homologous and isoforms of GSK-3. A two- to three-fold stimulation of the GS activity ratio above basal is produced when the inhibitor CHIR98014 is applied to insulin receptor-expressing CHO-IR cells or primary rat hepatocytes at increasing concentrations. For rat hepatocytes and CHO-IR, the concentration of CHIR-98014 that causes half-maximal GS stimulation (EC50) is 107 nM.[1]

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. Additionally, CHIR-98014 decreases the phosphorylation (Ser396) of tau protein in the cortex and hippocampus of postnatal rats.

Polypropylene 96-well plates are filled 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. In all cell-free assays, CHIR-98014 or controls are added to 3.5 μL of DMSO, followed by 50 μL of ATP stock to produce a final concentration of 1 μM ATP. Following incubation, triplicate 100-L aliquots are added to Combiplate 8 plates, which have 100-μL/well concentrations of 50 mM ATP and 20 mM EDTA. The wells are rinsed five times with PBS, filled with 200 L of scintillation fluid, sealed, and left for 30 minutes before being counted in a scintillation counter after the first hour. The entire process is carried out at room temperature.

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. Trypsinized cells are seeded in 6-well plates at a density of 1 × 106 cells/well in 2 mL of medium devoid of fetal bovine serum. After 24 hours, the medium is replaced with 1 mL of serum-free medium containing the GSK-3 inhibitor CHIR 98014 or control (final 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 fluoride, and 25 g/mL leupeptin (buffer A), and centrifuged for 15 min at 4 °C/14000 g. The GS activity in the absence is used to calculate the activity ratio for GS.

Drugs and drug administration[2]
SB216763 (30 mg kg−1) and CHIR98014 (30 mg kg−1) were re-suspended in DMSO and injected i.v. AR-A014418 (30 mg kg−1), was dissolved in 100% PEG400 and administered per os (p.o.) Indirubin-3′-monoxime (20 mg kg−1) and Alsterpaullone (20 mg kg−1) were dissolved in 20% DMSO/25% Tween-80 and injected i.p. and s.c., respectively. All drug studies were conducted using P12 rats from the same litter. Control animals were dosed with the respective vehicle and both groups were killed after 1, 2 and 4 h for brain exposure measurements (see the next section), western blotting and GSK-3β activity assays. Experiments measuring the efficacy of each compound were performed at least three times and at a time point determined by brain exposure data. LiCl (100 and 200 mg kg−1) was dissolved in sterile water, and administered p.o. to animals. P12 rats were killed 8 h after injection. Some of the littermates were used as the control group and dosed with NaCl (100 or 200 mg kg−1, p.o.) dissolved in sterile water.[2]

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Brain exposure measurements[2]
Rat brain homogenates were analysed for exposure levels of SB216763, Indirubin-3′-monoxime, Alsterpaullone, CHIR98014 and AR-A014418 using turbulent flow chromatography (HTLC) followed by detection by Tandem mass spectrometry (MS/MS). Four times 70% v w−1acetonitrile was added to the sample and homogenized in an autogizer robot. The brain homogenate was centrifuged at 6000 g for 15 min at 5 °C, and the supernatant was analysed. Calibration curves (1–1000 ng ml−1 brain homogenate) for each compound were prepared using brain homogenate from untreated rats. A total of 25 μl of 10% MeOH with internal standard (citalopram) was added to either 25 μl of brain homogenate or calibration standard, followed by centrifugation at 6000 g for 20 min at 5 °C). Ten microlitres of each sample was injected into the HTLC system using a HTS PAL autosampler. Samples with AR-A014418 were purified using 0.1% HCOOH in water for 15 s (2 ml min−1) using a Cyclone HTLC column (0.5 × 50 mm, 50 μm). The compounds were extracted from the HTLC using 100 μl 0.1% HCOOH/90% acetonitrile placed in the loop and transferred to the analytical column, X-Terra MS C8 (20 × 2.1 mm, 3.5 μm) with 0.1% HCOOH in water over 120 s (0.08 ml min−1) and eluted from the analytical column using a gradient from 0.1% HCOOH/2% MeCN to 0.1% HCOOH/98% acetonitrile for 45 s, followed by elution with 0.1% HCOOH/98% acetonitrile for 120 s flow 0.5 ml min−1). Detection of the compound was performed using Ultima triple-quadropole mass spectrometer (Waters) and positive ionization using multiple reaction monitoring set at optimal conditions. For AR-A014418 the transition 308.9 → 121.7 was used.

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Blood is obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose is measured directly or heparinized plasma is collected for measurement of glucose or insulin. Animals are prebled and randomly assigned to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals are fasted throughout the procedure with food removal early in the morning, 3 h before the first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). Food is taken away 16 hours prior to the test agent being administered when determining the time course of plasma glucose and insulin changes in fasting ZDF rats. The GS activity ratio is calculated as the GS activity in the presence of 5 mM glucose-6-phosphate divided by the activity in the absence of glucose-6-phosphate.

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

[2]. Br J Pharmacol. 2007 Nov;152(6):959-79.

CHIR-98014 is a member of the class of aminopyrimidines that is pyrimidine substituted by {2-[(6-amino-5-nitropyridin-2-yl)amino]ethyl}amino, 2,4-dichlorophenyl, and 1H-imidazol-1-yl groups at positions 2, 4 and 5, respectively. It is a potent ATP-competitive inhibitor of GSK3alpha and GSK3beta (IC50 values of 0.65 and 0.58 nM, respectively). It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor, an apoptosis inducer, an antineoplastic agent, a hypoglycemic agent, a Wnt signalling activator and a tau aggregation inhibitor. It is a secondary amino compound, a dichlorobenzene, a member of imidazoles, a diaminopyridine, an aminopyrimidine and a C-nitro compound.

C20H17CL2N9O2

486.3141

485.088

C, 49.39; H, 3.52; Cl, 14.58; N, 25.92; O, 6.58

252935-94-7

556813-39-9 (CHIR98024);252935-94-7 (CHIR98014);CHIR98014 HCl;

53396311

Light yellow to yellow solid powder

1.6±0.1 g/cm3

839.0±75.0 °C at 760 mmHg

461.2±37.1 °C

0.0±3.1 mmHg at 25°C

1.753

3.76

3

9

7

33

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([H])[H])N=1)[N+](=O)[O-])N1C([H])=NC([H])=C1[H])Cl

MDZCSIDIPDZWKL-UHFFFAOYSA-N

InChI=1S/C20H17Cl2N9O2/c21-12-1-2-13(14(22)9-12)18-16(30-8-7-24-11-30)10-27-20(29-18)26-6-5-25-17-4-3-15(31(32)33)19(23)28-17/h1-4,7-11H,5-6H2,(H3,23,25,28)(H,26,27,29)

6-N-[2-[[4-(2,4-dichlorophenyl)-5-imidazol-1-ylpyrimidin-2-yl]amino]ethyl]-3-nitropyridine-2,6-diamine

CT-98014; CT 98014; CT98014; CHIR 98014; CHIR-98014; 252935-94-7; 6-N-[2-[[4-(2,4-dichlorophenyl)-5-imidazol-1-ylpyrimidin-2-yl]amino]ethyl]-3-nitropyridine-2,6-diamine; CT-98014; 2,6-PYRIDINEDIAMINE, N6-[2-[[4-(2,4-DICHLOROPHENYL)-5-(1H-IMIDAZOL-1-YL)-2-PYRIMIDINYL]AMINO]ETHYL]-3-NITRO-; CHEMBL3185148; N2-(2-((4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-yl)amino)ethyl)-5-nitropyridine-2,6-diamine; N6-[2-[[4-(2,4-DICHLOROPHENYL)-5-(1H-IMIDAZOL-1-YL)-2-PYRIMIDINYL]AMINO]ETHYL]-3-NITRO-2,6-PYRIDINEDIAMINE; CHIR-98014; CHIR98014;CHIR98014 HCl; CHIR-98014 hydrochloride

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)

DMSO: ~8 mg/mL (~16.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

10%DMSO+90%corn oil: 1.1mg/mL (Please use freshly prepared in vivo formulations for optimal results.)