GSK-3 activity assay

GSK-3 kinase activity was measured, in the presence of various concentrations of SB 216763, in a reaction mixture containing final concentrations of 1 nM human GSK-3α, 50 mM MOPS pH 7.0, 0.2 mM EDTA, 10 mM Mg-acetate, 7.5 mM β-mercaptoethanol, 5% (w/v) glycerol, 0.01% (w/v) Tween-20, 10% (v/v) DMSO, and 28 μM GS-2 peptide substrate. The GS-2 peptide sequence corresponds to a region of glycogen synthase that was phosphorylated by GSK-3. The assay was initiated by the addition of 0.34 μCi [33P]γ-ATP. The total ATP concentration was 10 μM. Following 30 mins incubation at room temperature the assay was stopped by the addition of one third assay volume of 2.5% (v/v) H3PO4 containing 21 mM ATP. Samples were spotted onto P30 phosphocellulose mats and washed six times in 0.5% (v/v) H3PO4. The filter mats were sealed into sample bags containing Wallac betaplate scintillation fluid. 33P incorporation into the substrate peptide was determined by counting the mats in a Wallac microbeta scintillation counter.

Cell lines

Cerebellar granule neurons

Preparation method

The solubility of this compound in DMSO is >56.8mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 °C for several months.

Reaction Conditions

0.1 ~ 10 μM; 48 hrs

Applications

SB 216763 concentration-dependently reduced the death of cerebellar granule neurons induced by potassium-deprivation or LY-294002 treatment. 3 μM SB 216763 exhibited the maximal neuroprotection effect.

Animal models

Bleomycin (BLM)-induced lung inflammation and fibrosis mouse model

Dosage form

20 mg/kg; i.v.

Applications

In a Bleomycin (BLM)-induced lung inflammation and fibrosis mouse model, SB 216763 significantly prevented lung inflammation and subsequent fibrosis through significantly blocking the production of inflammatory cytokines MCP-1 and TNF-α by macrophages. Moreover, it significantly improved the survival, and markedly reduced alveolitis by inhibiting alveolar epithelial cell damage.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000 Oct;7(10):793-803.

[2]. Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, Reith AD. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 2001,77:94-102.

[3] Gurrieri C, Piazza F, Gnoato M, Montini B, Biasutto L, Gattazzo C, et al. 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), a glycogen synthase kinase-3 inhibitor, displays therapeutic properties in a mouse model of pulmonary inflammation and fibrosis. J Pharmacol Exp Ther 2010,332:785-794.