Thiamet G
目录号 : GC12736Thiamet G 是一种有效的 O-GlcNAcase 抑制剂 (Ki =21 nM),用于提高 O-GlcNAcylation 水平。
Cas No.:1009816-48-1
Sample solution is provided at 25 µL, 10mM.
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- Purity: >99.50%
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Cell experiment [1]: | |
Cell lines |
A549, HT29 and H1299 cell lines |
Preparation Method |
Cells were treated with 5 µM Thiamet-G for 24 h or the indicated time period. |
Reaction Conditions |
5µM for 24 hours |
Applications |
Thiamet G could markedly elevate the O-GlcNAcylation of A549, H1299 and HT29 cells. Thiamet-G treatment did not significantly affect the proliferation of A549, H1299 and HT29. Thiamet-G treatment significantly increased colony formation ability of A549, H1299 and HT29 cells. |
Animal experiment [2]: | |
Animal models |
Six-week-old male Sprague-Dawley rats |
Preparation Method |
For tau western blot study, rats were treated orally with thiamet-G, by inclusion of inhibitor in the drinking water at a dose of 200 mg kg-1 d-1. Animals were euthanized after one day of treatment. For the thiamet-G dose and time dependency study, six-week-old male Sprague-Dawley rats received single intravenous tail vein injections of either 2, 10 or 50 mg kg-1 and were euthanized at indicated times. |
Dosage form |
200 mg kg-1 d-1, oral˙2, 10 or 50 mg kg-1 d-1, intravenous injection. |
Applications |
Thiamet-G is orally bioavailable, thiamet-G can cross the blood brain barrier and inhibit O-GlcNAcase in vivo to cause increased O-GlcNAc levels in brain. |
References: [1]: Mi W, Gu Y, Han C, et al. O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy[J]. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2011, 1812(4): 514-519. |
Thiamet G, a potent inhibitor of O-GlcNAcase (Ki =21 nM) was used to increase O-GlcNAcylation levels [1]. Thiamet G is a stable compound whose fused thiazoline ring system geometrically mimics a transition state of the substrate-assisted enzymatic hydrolysis of protein-O-GlcNAc units and, in this way, effectively inhibits O-GlcNAcase function [2]. Thiamet-G is orally bioavailable, and thiamet-G can cross the blood brain barrier [1].
Thiamet G (5 μM, 24h) could markedly elevate the O-GlcNAcylation of human lung epithelial carcinoma A549, non-small cell lung carcinoma H1299 and colon tumor HT29 cells [3]. Thiamet G (25 μM, 24 h) treated PC-12 cells showed a gradual time-dependent increase in cellular O-GlcNAc levels that reached a maximum after approximately 12 h of exposure [1].
Thiamet G (2.5 μl of 35 μg/μl) dissolved in 0.9% NaCl was injected bilaterally into the lateral ventricles of the brains at a dose of 175 μg/mouse, RL2 positive bands showed a 5-fold increase in global O-GlcNAcylation 4.5 h after thiamet G injection and a 10-fold increase 24 h after injection [4]. Thiamet G (500 mg/kg/day p.o.) treatment was able to decrease the number of neurons showing tau pathology, decrease behavioral defects and reduce mice mortality in the Tau.P301L mouse model [5]. Thiamet-G treated Tau.P301L mice for 3 days in the drinking water (2.5 mg/ml) in the home-cage, improved their breathing deficit in normocapnia and in hypercapnia [6].
References:
[1]. Yuzwa S A, Macauley M S, Heinonen J E, et al. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo[J]. Nature chemical biology, 2008, 4(8): 483-490.
[2]. Fischer P M. Turning down tau phosphorylation[J]. Nature chemical biology, 2008, 4(8): 448-449.
[3]. Mi W, Gu Y, Han C, et al. O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy[J]. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2011, 1812(4): 514-519.
[4]. Yu Y, Zhang L, Li X, et al. Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation[J]. PloS one, 2012, 7(4): e35277.
[5]. Graham DL, Gray AJ, Joyce JA, Yu D, O'Moore J, Carlson GA, Shearman MS, Dellovade TL, Hering H. Increased O-GlcNAcylation reduces pathological tau without affecting its normal phosphorylation in a mouse model of tauopathy[J]. Neuropharmacology, 2014,1;79:307-13.
[6]. Borghgraef P, Menuet C, Theunis C, Louis JV, Devijver H, Maurin H, Smet-Nocca C, Lippens G, Hilaire G, Gijsen H, Moechars D. Increasing brain protein O-GlcNAc-ylation mitigates breathing defects and mortality of Tau. P301L mice[J]. PloS one, 2013, Dec 23;8(12):e84442.
Thiamet G 是一种有效的 O-GlcNAcase 抑制剂 (Ki =21 nM),用于提高 O-GlcNAcylation 水平[1]。 Thiamet G 是一种稳定的化合物,其稠合噻唑啉环系统在几何上模拟了蛋白质-O-GlcNAc 单元底物辅助酶水解的过渡态,从而有效抑制 O-GlcNAcase 功能[2]。 Thiamet-G 具有口服生物利用度,thiamet-G 可以穿过血脑屏障[1]。
Thiamet G (5 μM, 24h) 可显着提高人肺上皮癌 A549、非小细胞肺癌 H1299 和结肠肿瘤 HT29 细胞的 O-GlcNAcylation[3]。 Thiamet G(25 μM,24 小时)处理的 PC-12 细胞显示细胞 O-GlcNAc 水平随时间逐渐增加,在暴露约 12 小时后达到最大值[1]。
将溶解在 0.9% NaCl 中的 Thiamet G(2.5 μl,35 μg/μl)以 175 μg/小鼠的剂量双侧注射到大脑的侧脑室,RL2 阳性条带显示整体 O2 增加了 5 倍-Thiamet G 注射后 4.5 小时的 GlcNAcylation,注射后 24 小时增加 10 倍[4]。在 Tau.P301L 小鼠模型中,Thiamet G(500 mg/kg/天口服)处理能够减少显示 tau 病理的神经元数量,减少行为缺陷并降低小鼠死亡率[5]。 Thiamet-G 在饲养笼中用饮用水 (2.5 mg/ml) 处理 Tau.P301L 小鼠 3 天,改善了正常碳酸血症和高碳酸血症时的呼吸缺陷[6]。
Cas No. | 1009816-48-1 | SDF | |
化学名 | 2-(ethylamino)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d][1,3]thiazole-6,7-diol | ||
Canonical SMILES | CCNC1=NC2C(C(C(OC2S1)CO)O)O | ||
分子式 | C9H16N2O4S | 分子量 | 248.3 |
溶解度 | ≥ 12.4mg/mL in DMSO, ≥ 100mg/mL in Water | 储存条件 | Store at -20°C |
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Shipping Condition | 评估样品解决方案:配备蓝冰进行发货。所有其他可用尺寸:配备RT,或根据请求配备蓝冰。 |
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1 mg | 5 mg | 10 mg | |
1 mM | 4.0274 mL | 20.1369 mL | 40.2739 mL |
5 mM | 0.8055 mL | 4.0274 mL | 8.0548 mL |
10 mM | 0.4027 mL | 2.0137 mL | 4.0274 mL |
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Thiamet-G-mediated inhibition of O-GlcNAcase sensitizes human leukemia cells to microtubule-stabilizing agent paclitaxel
Although the microtubule-stabilizing agent paclitaxel has been widely used for treatment of several cancer types, particularly for the malignancies of epithelia origin, it only shows limited efficacy on hematological malignancies. Emerging roles of O-GlcNAcylation modification of proteins in various cancer types have implicated the key enzymes catalyzing this reversible modification as targets for cancer therapy. Here, we show that the highly selective O-GlcNAcase (OGA) inhibitor thiamet-G significantly sensitized human leukemia cell lines to paclitaxel, with an approximate 10-fold leftward shift of IC50. Knockdown of OGA by siRNAs or inhibition of OGA by thiamet-G did not influence the cell viability. Furthermore, we demonstrated that thiamet-G binds to OGA in competition with 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate, an analogue of O-GlcNAc UDP, thereby suppressing the activity of OGA. Importantly, inhibition of OGA by thiamet-G decreased the phosphorylation of microtubule-associated protein Tau and caused alterations of microtubule network in cells. It is noteworthy that paclitaxel combined with thiamet-G resulted in more profound perturbations on microtubule stability than did either one alone, which may implicate the underlying mechanism of thiamet-G-mediated sensitization of leukemia cells to paclitaxel. These findings thus suggest that a regimen of paclitaxel combined with OGA inhibitor might be more effective for the treatment of human leukemia.
O-GlcNAcylation of light chain serine 12 mediates rituximab production doubled by thiamet G
O-Glycosylation occurs in recombinant proteins produced by CHO cells, but this phenomenon has not been studied extensively. Here, we report that rituximab is an O-linked N-acetyl-glucosaminylated (O-GlcNAcylated) protein and the production of rituximab is increased by thiamet G, an inhibitor of O-GlcNAcase. The production of rituximab doubled with OGA inhibition and decreased with O-GlcNAc transferase inhibition. O-GlcNAc-specific antibody and metabolic labelling with azidO-GlcNAc confirmed the increased O-GlcNAcylation with thiamet G. Protein mass analysis revealed that serine 7, 12, and 14 of the rituximab light chain were O-GlcNAcylated. S12A mutation of the light chain decreased rituximab stability and failed to increase the production with thiamet G without any significant changes of mRNA level. Cytotoxicity and thermal stability assays confirmed that there were no differences in the biological and physical properties of rituximab produced by thiamet G treatment. Therefore, thiamet G treatment improves the production of rituximab without significantly altering its function.
Osteoclast Differentiation Is Suppressed by Increased O-GlcNAcylation Due to Thiamet G Treatment
Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.
Thiamet G mediates neuroprotection in experimental stroke by modulating microglia/macrophage polarization and inhibiting NF-κB p65 signaling
Inflammatory responses are accountable for secondary injury induced by acute ischemic stroke (AIS). Previous studies indicated that O-GlcNAc modification (O-GlcNAcylation) is involved in the pathology of AIS, and increase of O-GlcNAcylation by glucosamine attenuated the brain damage after ischemia/reperfusion. Inhibition of β-N-acetylglucosaminidase (OGA) with thiamet G (TMG) is an alternative option for accumulating O-GlcNAcylated proteins. In this study, we investigate the neuroprotective effect of TMG in a mouse model of experimental stroke. Our results indicate that TMG administration either before or after middle cerebral artery occlusion (MCAO) surgery dramatically reduced infarct volume compared with that in untreated controls. TMG treatment ameliorated the neurological deficits and improved clinical outcomes in neurobehavioral tests by modulating the expression of pro-inflammatory and anti-inflammatory cytokines. Additionally, TMG administration reduced the number of Iba1+ cells in MCAO mice, decreased expression of the M1 markers, and increased expression of the M2 markers in vivo. In vitro, M1 polarization of BV2 cells was inhibited by TMG treatment. Moreover, TMG decreased the expression of iNOS and COX2 mainly by suppressing NF-κB p65 signaling. These results suggest that TMG exerts a neuroprotective effect and could be useful as an anti-inflammatory agent for ischemic stroke therapy.
Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes
Rationale: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality.
Objective: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII.
Methods and results: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation.
Conclusions: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.