Background
SB-415286 is a potent and selective cell permeable inhibitor of glycogen synthase kinase-3 (GSK-3) with Ki of 31 nM. It shows similar potency against GSK-3 and GSK3β [1].
SB-415286 inhibited GSK-3 activity and promoted glycogen synthesis in human liver cells and induced expression of reporter gene regulated by catenin-LEF/TCF in HEK293 cells [1]. In primary neurons, it can prevent cell death induced by repressed PI3k pathway activity [2]. Further studies showed that reduced GSK3β activity induced by SB-415286 could inhibit down-regulation of cyclin D1, cell cycle arrest and chemosensitivity, which were all mediated by rapamycin [3]. Pharmacologic inhibition of GSK-3β dramatically impaired p53-dependent transactivation of p21 and Puma but facilitated p53-dependent conformational activation of Bax, resulting in the conversion of p53-mediated damage response from cell cycle arrest to apoptosis [4]. SB-415286 reduced ischemia-reperfusion injury by mechanisms which were associated with mitochondria. SB-415286 reduced adenine nucleotide transport and phosphorylation of VDAC, then increased Bcl-2 binding to mitochondria and blocked opening of the mitochondrial permeability transition pore in cardiomyocytes [5]. SB-415286 had protective effect of hippocampal neurons on radiation-induced apoptosis as well. GSK-3β inhibition induced by SB-415286 could result in the upregulation of MDM2, which, in turn, regulated p53 degradation and p53-dependent cellular responses [6].
Recent research in a mouse model further confirmed that SB-415286 is a neuroprotectant against radiation-induced central nervous system necrosis. Mice treated with SB415286 prior to irradiation (i.e. a single 45-Gy fraction targeted to the left hemisphere), showed significant protection from radiation-induced necrosis, which was determined by in vivo MRI, in contrast with DMSO-treated mice [7].
参考文献:
Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.? Chem Biol. 2000 Oct;7(10):793-803.
Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death.? J Neurochem. 2001 Apr;77(1):94-102.
Role of glycogen synthase kinase 3beta in rapamycin-mediated cell cycle regulation and chemosensitivity.? Cancer Res. 2005 Mar 1;65(5):1961-72.
Pharmacologic modulation of glycogen synthase kinase-3beta promotes p53-dependent apoptosis through a direct Bax-mediated mitochondrial pathway in colorectal cancer cells.? Cancer Res. 2005 Oct 1;65(19):9012-20.
Glycogen synthase kinase 3 inhibition slows mitochondrial adenine nucleotide transport and regulates voltage-dependent anion channel phosphorylation.? Circ Res. 2008 Oct 24;103(9):983-91. doi: 10.1161/CIRCRESAHA.108.178970. Epub 2008 Sep 18.
Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway.? Cell Death Differ. 2012 Mar;19(3):387-96. doi: 10.1038/cdd.2011.94. Epub 2011 Jul 8.
A GSK-3β inhibitor protects against radiation necrosis in mouse brain.? Int J Radiat Oncol Biol Phys. 2014 Jul 15;89(4):714-21. doi: 10.1016/j.ijrobp.2014.04.018.
Protocol
Kinase experiment: | GSK-3 kinase activity is measured, in the presence or absence of SB-216763 or SB-415286, 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. The assay is 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 is 10 μM (IC50 determinations) or ranges from 0 to 45 μM (Ki determinations). Following 30 min incubation at room temperature the assay is stopped by the addition of one third assay volume of 2.5% (v/v) H3PO4 containing 21 mM ATP. Samples are spotted onto P30 phosphocellulose mats and these are washed six times in 0.5% (v/v) H3PO4. The filter mats are sealed into sample bags containing Wallac betaplate scintillation fluid. 33P incorporation into the substrate peptide is determined by counting the mats in a Wallac microbeta scintillation counter[1]. |
Cell experiment: | B65 cells are used after 24 h of in vitro culture. CGN are used after 7-8 days in vitro. Lithium and SB-415286 are dissolved in culture media and DMSO, respectively, and added to the neuronal preparation at the precise concentrations, 1 h before addition H2O2 (50 μM to 1 mM). To assess the loss in cell viability, we use the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] method. MTT is added to the cells at a final concentration of 250 μM and incubated for 1 h, allowing the reduction in MTT to produce a dark blue formazan product. Media are then removed, and cells are dissolved in dimethylsulfoxide. Formazan production is measured by the absorbency change at 595 nm using a microplate reader. Viability results are expressed as percentages. The absorbency measured from non-treated cells is taken to be 100%[2]. |
参考文献: [1]. Coghlan MP, 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.
[2]. Pizarro JG, et al. Neuroprotective effects of SB-415286 on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons. Int J Dev Neurosci. 2008 May-Jun;26(3-4):269-76.
[3]. MacAulay K, et al. Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase. Eur J Biochem. 2003 Sep;270(18):3829-38. |
没有评价数据