SB 216763

Enzyme inhibition assay^[1]:

Preparation method

GSK-3 kinase activity was measured, in the presence or absence of SB 216763, in a reaction mixture containing final concentrations of: 1 nM human GSK-3α or rabbit GSK3α; 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; 28 μM GS-2 peptide substrate.

The GS-2 peptide sequence corresponds to a region of glycogen synthase that is phosphorylated by GSK-3 as previously described.

The assay was initiated by the addition of 0.34 μCi ^[33P]γ-ATP (IC₅₀ determinations) or 2.7 μCi ^[33P]γ-ATP (Ki determinations).

The total ATP concentration was 10 μM (IC₅₀ determinations) or ranged from 0 to 45 μM (K_i determinations). Following 30 min 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 these were 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.

Applications

SB 216763 inhibited human GSK-3α with IC₅₀ of 34 nM, when assayed in the presence of 0.01 mM ATP.

Cell experiment^[2]:

Cell lines

HEK293 cells

Preparation method

Cells were electroporated with the Super TOPFlash and pGL4.73 Renilla luciferase reporters. The cells were treated in quadruplicate wells with a vehicle control (0.1% DMSO) or the indicated doses of SB 216763 for 24 hours.

Reaction Conditions

1–20 µM SB 216763；24 hours

Applications

SB 216763 induces β-catenin mediated-transcription in a dose-dependent manner.

Animal experiment^[3]:

Animal models

Adult male Wistar rats

Preparation method

The animals were divided into four groups (n = 9/group): (1) Vehicle infusion group treated with vehicle alone; (2) Aldo-salt group treated with an infusion of Aldo-salt (1 mg/kg/day diluted in sunflower oil and administered by subcutaneous injection); (3) Aldo-salt plus SB 216763 group treated with an infusion of Aldo-salt plus SB 216763 at 1.5 mg/kg/day; and (4) SB 216763 group.

Dosage form

1-1.5 mg/kg/day；4weeks；s.c

Applications

SB 216763 suppresses cardiac inflammation and fibrosis caused by Aldo-salt.

References:

[1].  Coghlan MP, Culbert AA, et,al. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000 Oct;7(10):793-803. doi: 10.1016/s1074-5521(00)00025-9. PMID: 11033082.

[2]. Kirby LA, Schott JT, et,al. Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells. PLoS One. 2012;7(6):e39329. doi: 10.1371/journal.pone.0039329. Epub 2012 Jun 26. PMID: 22745733; PMCID: PMC3383737.

[3].Zhang YD, Ding XJ, et,al.SB-216763, a GSK-3β inhibitor, protects against aldosterone-induced cardiac, and renal injury by activating autophagy. J Cell Biochem. 2018 Jul;119(7):5934-5943. doi: 10.1002/jcb.26788. Epub 2018 Mar 30. PMID: 29600538; PMCID: PMC6001754.