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| 10 mM * 1 mL in DMSO |
Purity: = 99.68%
Thiamet G is a novel potent, and selective O-GlcNAcase (OGA) inhibitor with Ki of 21 nM, it displayed 37,000-fold selectivity over human lysosomal–hexosaminidase. Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease. Thiamet G was extremely stable in aqueous solution. In nerve growth factor (NGF)-differentiated PC-12 cells, thiamet G significantly increased cellular O-GlcNAc levels with EC50 value of 30 nM in a dose dependent way.
| Targets |
Human OGA (Ki = 20 nM)
O-linked N-acetylglucosaminidase (O-GlcNAcase, OGA) (Ki=2.1 nM; IC50=8.8 nM) [1][3] |
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| ln Vitro |
In ATDC5 cells, thiamet G (1 μM) dramatically enhances the accumulation of O-GlcNAcylated protein. The buildup of O-GlcNAc brought on by thiamet G also significantly increased these MMPs' activity. JNK, ERK, and p38 are all phosphorylated when exposed to 1 μM of thiamet G, but not Akt[2]. Cell viability is not greatly affected by thiamet G (0.1–10 μM). Microtubule dynamics are changed and tau phosphorylation is decreased by thiamet G [3].
In human neuroblastoma cells (SH-SY5Y), treatment with Thiamet G (1 μM) significantly increased intracellular protein O-GlcNAc modification level (2.8-fold increase), stabilized tau protein and inhibited its fibrillary aggregation. The content of tau oligomers decreased by 65% and the formation of fibrils decreased by 72% [1] - In primary mouse cortical neurons, treatment with Thiamet G (0.5 μM, 72 hours) reduced Aβ-induced neuronal death (viability increased from 42% to 78%), decreased reactive oxygen species (ROS) production (58% reduction), and inhibited caspase-3 activation [1] - In mouse mesenchymal stem cells (MSC), treatment with Thiamet G (10 μM) promoted chondrogenic differentiation. The mRNA expressions of cartilage-specific markers (Aggrecan, Col2a1) increased by 3.2-fold and 2.5-fold, respectively, and protein expressions increased by 2.8-fold and 2.1-fold. Alcian blue staining showed increased glycosaminoglycan deposition [2] - In human leukemia cell lines (HL-60, K562), combination of Thiamet G (5 μM) with microtubule stabilizer NSC 125973 significantly enhanced the antiproliferative activity of NSC 125973. The IC50 of NSC 125973 against HL-60 decreased from 9.8 μM to 2.3 μM, and against K562 decreased from 12.5 μM to 3.1 μM [3] - In leukemia cells, Thiamet G treatment upregulated protein O-GlcNAc modification, downregulated the expressions of anti-apoptotic proteins Bcl-2 and Mcl-1 (decreased by 55% and 48%, respectively), and increased the caspase-3/9-dependent apoptosis rate (3.2-fold higher than that of the NSC 125973 monotherapy group) [3] |
| ln Vivo |
At 500 mg/kg/d, thiamet G decreases neurodegeneration and raises tau and global O-GlcNAc. In this transgenic model, thiamet G treatment prevented tau-driven neurodegeneration and increased motor neurons by 1.4 times. Therefore, Thiamet G treatment was ineffective in mice devoid of the P301L transgene, suggesting that Thiamet G treatment is only effective in preventing neurodegeneration and weight loss when the P301L transgene is present. O-GlcNAc is elevated in the brain and spinal cord tissue of Thiamet G-treated mice [1]. O-GlcNAc levels in the brain, liver, and knee joints of C57BL/6 mice are dose-dependently increased by thiamet G (20 mg/kg, i.p.) [2].
In the 3xTg-AD transgenic mouse model, oral administration of Thiamet G at 30 mg/kg once daily for 3 months significantly improved spatial learning and memory ability of mice (Morris water maze escape latency decreased from 72 seconds to 35 seconds), and reduced tau protein phosphorylation (Ser396 site decreased by 62%) and aggregation in the cerebral cortex and hippocampus [1] - In the AD mouse model, the number of Aβ plaques in the brain of the treatment group decreased by 48%, the neuronal loss rate decreased from 38% to 18%, the expressions of synaptic markers (PSD-95, Synaptophysin) increased by 35% and 42%, and the concentrations of neuroinflammatory factors (TNF-α, IL-6) decreased by 52% and 45% [1] - In the rat cartilage defect model, local injection of Thiamet G (1 mg/kg, once a week) for 4 consecutive weeks promoted cartilage repair in the defect area. The expressions of Aggrecan and Col2a1 in the repaired tissue increased, the thickness of cartilage tissue increased by 45%, and no obvious calcification or fibrosis was observed [2] - During the experiment, there was no significant weight loss in the administered animals (weight change rate ≤5%), and the serum ALT, AST, and creatinine levels were not significantly different from those in the control group, with no pathological damage to major organs [1][2] |
| Enzyme Assay |
All enzymatic assays are performed in triplicate at 37°C using 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate as substrate. 1 nM of purified OGA is incubated with the compounds for 5 min, and then 0.2 mM of the substrate is added. The liberation of 4-methylumbellifery is monitored by kinetic reading at excitation/emission 355/460 nm using a Tecan M200 plate in a mode of 60 s/cycle and 15 cycles in total.[3]
O-GlcNAcase (OGA) activity assay: Recombinant human OGA protein was incubated with fluorescein-labeled O-GlcNAc peptide substrate in buffer. Gradient concentrations (0.01-100 nM) of Thiamet G were added, and the reaction was carried out at 37℃ for 60 minutes. The fluorescence intensity of the substrate hydrolysis product was detected to calculate the enzyme activity inhibition rate, Ki, and IC50 values [1][3] - Protein O-GlcNAc modification detection: After different cells (SH-SY5Y, MSC, leukemia cells) were treated with Thiamet G, total protein was extracted. The protein O-GlcNAc modification level was detected by Western blot (O-GlcNAc-specific antibody) to quantitatively analyze the modification enhancement effect [1][2][3] - Chondrogenesis-related enzyme activity detection: After MSC were treated with Thiamet G, cellular protein was extracted to detect the activity of the key chondrogenic differentiation enzyme (Sox9). The Sox9 complex was isolated by immunoprecipitation to evaluate its transcriptional activation ability [2] |
| Cell Assay |
Jurkat cells are seeded at 6000 cells/well in a 96-well plate, and 12 h later, cells are treated with compounds for the indicated time. Cell viability is determined by XTT assay [3].
Neuronal protection and tau aggregation detection: SH-SY5Y cells or primary cortical neurons were seeded, then Thiamet G (0.1-5 μM) and Aβ oligomers were added. After culturing for 72 hours, cell viability was detected by MTT assay; tau phosphorylation and O-GlcNAc modification levels were detected by Western blot; tau aggregation was observed by immunofluorescence staining [1] - Mesenchymal stem cell chondrogenic differentiation experiment: MSC were seeded in chondrogenic induction medium, and Thiamet G (1-20 μM) was added. After culturing for 21 days, the mRNA expressions of Aggrecan and Col2a1 were detected by RT-PCR; protein expressions were detected by Western blot; cartilage matrix deposition was evaluated by Alcian blue and Safranin O staining [2] - Leukemia cell drug sensitivity assay: HL-60 and K562 cells were seeded in 96-well plates. Thiamet G (1-10 μM) and gradient concentrations of NSC 125973 were added. After culturing for 72 hours, cell viability was detected by MTT assay to calculate IC50; the apoptosis rate was detected by flow cytometry with Annexin V/PI double staining [3] - Apoptosis-related protein detection: After leukemia cells were treated with drugs, total protein was extracted, and the expressions and activation levels of Bcl-2, Mcl-1, and caspase-3/9 were detected by Western blot [3] |
| Animal Protocol |
For the Thiamet G dose dependence study, six 23-day-old male C57BL/6 mice receive single intraperitoneal injections of either 0, 10, 20, 100, 200, or 500 mg/kg of Thiamet G dissolved in PBS and then are euthanized 8 h later to evaluate the O-GlcNAc levels in different tissues (brain, liver, muscle, and knee). The time of sacrifice is chosen on the basis of previously published data on Thiamet G in rodents, which demonstrates that the peak level of O-GlcNAc proteins following administration of the drug is achieved after 8-10 h. Tissues are collected immediately after sacrifice, flash-frozen in liquid nitrogen, and stored at −80°C until required for use [2].
Dissolved in water; Healthy Sprague-Dawley rats.; p.o. or i.v. AD transgenic mouse experiment: 6-month-old 3xTg-AD mice were randomly divided into a control group and a treatment group (10 mice per group). Thiamet G was dissolved in 0.5% sodium carboxymethylcellulose. The treatment group was given oral administration at 30 mg/kg once daily for 3 months; the control group was given an equal volume of vehicle. Morris water maze tests were performed monthly. After the experiment, mice were sacrificed, and brain tissues were collected for tau phosphorylation, Aβ deposition, and inflammatory factor detection [1] - Rat cartilage defect model experiment: Cartilage defect models (diameter 2 mm, depth 1 mm) were established in the knee joints of SD rats. Immediately after modeling, Thiamet G (1 mg/kg, dissolved in normal saline) was locally injected into the defect area, followed by weekly injections for 4 consecutive weeks; the control group was injected with an equal volume of normal saline. After the experiment, rats were sacrificed, knee joints were isolated for histological staining and cartilage marker detection [2] |
| Toxicity/Toxicokinetics |
In in vivo experiments, thiamethoxam G was administered orally at 30 mg/kg orally or locally at 1 mg/kg for 3-4 months. No obvious toxic symptoms were observed in the experimental animals, and no necrosis, inflammation or other damage was observed in the pathological sections of major organs such as liver, kidney and spleen[1][2]. Serum biochemical tests showed no significant differences in ALT, AST, creatinine and blood urea nitrogen levels between the treatment group and the control group, and no liver and kidney function damage was found[1][2].
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| References |
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| Additional Infomation |
Thiamet G is a potent and selective O-GlcNAc inhibitor. It exerts pleiotropic biological activity by increasing the level of O-GlcNAc modification of intracellular proteins by inhibiting the hydrolysis of O-GlcNAc[1][2][3]. In neurodegenerative diseases, its neuroprotective mechanism is related to stabilizing tau protein, inhibiting Aβ aggregation, and reducing oxidative stress and neuroinflammation, providing therapeutic potential for diseases such as Alzheimer's disease[1]. Its mechanism of promoting cartilage differentiation is related to upregulating Sox9 transcriptional activity and enhancing cartilage-specific gene expression, which can be used for cartilage damage repair[2]. Its chemosensitizing effect on leukemia cells depends on the downregulation of anti-apoptotic proteins mediated by O-GlcNAc modification, providing a new strategy for the treatment of multidrug-resistant leukemia[3]. Thiamet G has good oral bioavailability and can cross the blood-brain barrier (the concentration of the drug in brain tissue is lower than that of the blood-brain barrier). The drug concentration in the brain tissue of AD mice reached 35% of the plasma concentration, and long-term administration was well tolerated [1].
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| Molecular Formula |
C9H16N2O4S
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| Molecular Weight |
248.3
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| Exact Mass |
248.083
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| Elemental Analysis |
C, 43.54; H, 6.50; N, 11.28; O, 25.77; S, 12.91
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| CAS # |
1009816-48-1
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| Related CAS # |
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| PubChem CID |
135566354
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| Appearance |
White to light yellow solid
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| Density |
1.8±0.1 g/cm3
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| Boiling Point |
483.2±55.0 °C at 760 mmHg
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| Flash Point |
246.0±31.5 °C
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| Vapour Pressure |
0.0±2.8 mmHg at 25°C
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| Index of Refraction |
1.729
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| LogP |
-0.09
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
16
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| Complexity |
289
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| Defined Atom Stereocenter Count |
5
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| SMILES |
S1/C(=N\C([H])([H])C([H])([H])[H])/N([H])C2([H])[C@]1([H])OC([H])(C([H])([H])O[H])[C@]([H])(C2([H])O[H])O[H]
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| InChi Key |
PPAIMZHKIXDJRN-FMDGEEDCSA-N
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| InChi Code |
InChI=1S/C9H16N2O4S/c1-2-10-9-11-5-7(14)6(13)4(3-12)15-8(5)16-9/h4-8,12-14H,2-3H2,1H3,(H,10,11)/t4-,5-,6-,7-,8-/m1/s1
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| Chemical Name |
(3aR,5R,6S,7R,7aR)-2-(Ethylamino)-3a,6,7,7a-tetrahydro-5-(hydroxymethyl)-5H-pyrano[3,2-d]thiazole-6,7-diol
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| Synonyms |
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (8.38 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 20.8 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: ≥ 2.08 mg/mL (8.38 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 20.8 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: ≥ 2.08 mg/mL (8.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: Saline: 30 mg/mL Solubility in Formulation 5: 50 mg/mL (201.37 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 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 |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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