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

GSK-3 is more than 500-fold more selective for CHIR-99021 than its nearest homologs, CDC2 and ERK2, as well as other protein kinases. In addition, CHIR-99021 exhibits only modest inhibition of 23 nonkinase enzymes and only weak binding to a panel of 22 pharmacologically significant receptors. With an EC50 of 0.763 μM, CHIR-99021 causes the activation of glycogen synthase (GS) in CHO-IR cells that express the insulin receptor. [1] In addition to mimicking the effects of insulin, CHIR-99021's (3 μM) inhibition of GSK-3 causes an increase in free cytosolic -catenin by 1.9-fold, simulating the canonical Wnt signaling pathway in 3T3-L1 preadipocytes. By preventing the induction of CCAAT/enhancer-binding protein (C/EBP) and peroxisome proliferator-activated receptor γ (PPARγ) during any of the first three days of differentiation, CHIR-99021 treatment prevents preadipocyte differentiation with an IC50 of 0.3 μM. [2] Unlike lithium chloride and AR-A014418, CHIR-99021 treatment does not reduce the viability of INS-1E cells even at high concentrations. Instead, CHIR-99021 significantly reduces the rate of INS-1E cell death brought on by high glucose and high palmitate in a concentration-dependent manner. It also increases the rate of INS-1E cell proliferation robustly and dose-dependently. At concentrations as low as 1 μM, CHIR-99021 stimulates primary beta cell replication in isolated rat islets, with a 2-3 fold increase in cell replication after treatment with 5 μM of CHIR-99021.[3]

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]

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 27 nM GSK-3α or 29 nM GSK-3β, and 0.5 μM biotin-CREB peptide substrate. In all cell-free assays, different CHIR-99021 concentrations are added to 3.5 μL of DMSO before 50 μL of ATP stock to produce a final concentration of 1 M ATP. Following incubation, triplicate 100-μL aliquots are added to Combiplate 8 plates, which have 100-μL/well concentrations of 50 mM ATP and 20 mM EDTA. After 1 hour, the wells are rinsed five times with PBS, filled with 200 μL of scintillation fluid, sealed, left 30 minutes, and counted in a scintillation counter. All steps are performed at room temperature.

---

Kinases and kinase assays.[1]
Erk2, protein kinase C (PKC)-α, PKC-ζ, p90RSK2, c-src, AMPK, and pdk1 kinases were purchased from Upstate Biotechnology. DNA-PK was purified from HeLa cells as described previously. Other recombinant human protein kinases were expressed in SF9 cells with “glu” or hexahis peptide tags. Glu-tagged proteins were purified as described previously, and his-tagged proteins were purified according to the manufacturer’s instructions. 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; 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; see 33); 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 (see 31); 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 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).

---

Enzyme and receptor panels.[1]
Selectivity against nonkinase enzymes was tested on the Cerep “Enzyme” panel, including acetylcholinesterase; adenylate cyclase; Na/K ATPase; cathepsin B and G; cyclooxygenase 1 and 2; ECE; epithelial growth factor receptor; elastase; guanylate cyclase; HIV-1 protease; inducible nitric oxide synthase; 5-lipoxygenase; monoamine oxidase A and B; phosphodiesterase I, II, III, and IV; PKC; phospholipase A2 and C; and tyrosine hydroxylase. Selectivity against receptors was tested on the MDS “Profiling” panel, including adenosine A1; adrenergic (α1 and α2 nonselective and β1 and β2); calcium channel type L; dopamine D1 and D2; estrogen α; GABAA (agonist site and sodium channel); glucocorticoid; glutamate (NMDA/phencyclidine and nonselective); glycine (strychnine sensitive); histamine H1 (central); insulin; muscarinic M2 and M3; opiate δ, κ, and μ; phorbol ester; potassium channel; progesterone; serotonin (5-HT1 and 5-HT2/nonselective); sigma (nonselective); sodium channel (site 2); and testosterone.

---

GS activity assays.[1]
CHO-IR cells expressing human insulin receptor, were grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. 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. Primary hepatocytes from male Sprague Dawley rats that weighed <140 g were prepared at the Rice Liver Laboratory and used 1–3 h after isolation. Aliquots of 1 × 106 cells in 1 ml of DMEM/F12 medium plus 0.2% BSA and GSK-3 inhibitors or controls were 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 was again performed using the method of Thomas et al.

Cells are maintained for 24 hours in starvation medium (culture medium with only 5 mM glucose, 1% fetal calf serum). Then,cells are exposed to CHIR-99021 at various concentrations for 1, 3, or 4 days. Cellular DNA is stained with CyQuant dye, which turns fluorescent when bound to DNA, in order to count the number of cells present. Using the FLUOstar Optima reader, fluorescence is measured after 30 minutes of incubation. BrdUrd incorporation controls cell replication. Before the cells are fixed with FixDenat solution and incubated with monoclonal anti-BrdUrd-POD antibodies, BrdUrd labeling solution is added to the medium for the final four hours of the experiment. The light emission is measured in a microplate luminometer using the Analyst HT detection system after substrate solution is added to each well.

Female db/db mice or male ZDF rats with type 2 diabetes
~48 mg/kg
Oral Administration

---

Efficacy models.[1]
Blood was obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose was measured directly or heparinized plasma was collected for measurement of glucose or insulin. Animals were prebled and randomized to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals were fasted throughout the procedure with food removal early in the morning, 3 h before first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). When the time course of plasma glucose and insulin changes in fasting ZDF rats was measured, food was removed ∼16 h before test agent administration. The glucose challenges in the GTT were 1.35 g/kg i.p. (ipGTT) or 2 g/kg via oral gavage (oGTT). Test inhibitors were formulated as solutions in 20 mmol/l citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose.

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

[2]. J Biol Chem. 2002 Aug 23;277(34):30998-1004.

[3]. J Biol Chem. 2007 Apr 20;282(16):12030-7.

Insulin resistance plays a central role in the development of type 2 diabetes, but the precise defects in insulin action remain to be elucidated. Glycogen synthase kinase 3 (GSK-3) can negatively regulate several aspects of insulin signaling, and elevated levels of GSK-3 have been reported in skeletal muscle from diabetic rodents and humans. A limited amount of information is available regarding the utility of highly selective inhibitors of GSK-3 for the modification of insulin action under conditions of insulin resistance. In the present investigation, we describe novel substituted aminopyrimidine derivatives that inhibit human GSK-3 potently (K(i) < 10 nmol/l) with at least 500-fold selectivity against 20 other protein kinases. These low molecular weight compounds activated glycogen synthase at approximately 100 nmol/l in cultured CHO cells transfected with the insulin receptor and in primary hepatocytes isolated from Sprague-Dawley rats, and at 500 nmol/l in isolated type 1 skeletal muscle of both lean Zucker and ZDF rats. It is interesting that these GSK-3 inhibitors enhanced insulin-stimulated glucose transport in type 1 skeletal muscle from the insulin-resistant ZDF rats but not from insulin-sensitive lean Zucker rats. Single oral or subcutaneous doses of the inhibitors (30-48 mg/kg) rapidly lowered blood glucose levels and improved glucose disposal after oral or intravenous glucose challenges in ZDF rats and db/db mice, without causing hypoglycemia or markedly elevating insulin. Collectively, our results suggest that these selective GSK-3 inhibitors may be useful as acute-acting therapeutics for the treatment of the insulin resistance of type 2 diabetes.[1]

---

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.[2]

---

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.[3]

C22H19CL3N8

501.7989

500.079

C, 52.66; H, 3.82; Cl, 21.19; N, 22.33

1797989-42-4

Laduviglusib;252917-06-9;Laduviglusib trihydrochloride;1782235-14-6

71295844

White to yellow solid powder

0

4

7

7

33

645

0

ClC1C=C(C=CC=1C1C(=CN=C(N=1)NCCNC1C=CC(C#N)=CN=1)C1=NC=C(C)N1)Cl.Cl

SCQDMKUZHIGAIB-UHFFFAOYSA-N

InChI=1S/C22H18Cl2N8.ClH/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);1H

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

GSK 3IXV; CHIR99021; CHIR 99021; CHIR-911; CHIR911; CHIR 911; CT- 99021; GSK 3 inhibitor XVI; CT-99021; CT- 99021; CHIR-73911; CHIR73911; CHIR 73911

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~100 mg/mL (~199.3 mM)
Water: ~1.5 mg/mL(~3 mM)
Ethanol: ~100 mg/mL(~199.3 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (5.98 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 30.0 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: ≥ 3 mg/mL (5.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 3 mg/mL (5.98 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: ~20 mg/mL (40 mM) in 5% DMSO+30% PEG 300+ddH2O, clear solution
Solubility in Formulation 5: ~5 mg/mL (10 mM) in PBS, clear solution
Solubility in Formulation 6: ≥ 3 mg/mL (6 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 30 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 7: ≥ 3 mg/mL (6 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline), clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 30 mg/mL of DMSO stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well (clear solution).
Preparation of 20% SBE-β-CD in Saline (（4°C，1 week）): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Solubility in Formulation 8: ≥ 3 mg/mL (6 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 30 mg/mL of DMSO stock solution and add to 900 μL of corn oil, mix well (clear solution).

---

Solubility in Formulation 9: 5 mg/mL (9.96 mM) in PBS (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.)