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| Other Sizes |
| Targets |
Proteasome (26S and 20S core particle) – alloxan inhibits the chymotrypsin-like and trypsin-like peptidase activities. [1]
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| ln Vitro |
Purified 26S and 20S proteasomes' peptidase activity is inhibited by alloxan hydrate [1].
In NRK cells treated with 5 mM alloxan overnight, alloxan caused a significant accumulation of ubiquitinated proteins, as shown by anti-ubiquitin Western blot of the SDS-solubilized pellet fractions. [1] - In 293 cells transfected with GFP-CL1 (a proteasome-specific reporter), treatment with 10 mM alloxan plus 50 μg/ml cycloheximide significantly reduced the clearance rate of GFP-CL1 compared to control, indicating impaired ubiquitin-proteasome system function. [1] - NRK cells treated with 5 mM alloxan for 24 hr showed significant inhibition of both chymotrypsin-like (using suc-LLVY-AMC) and trypsin-like (using Boc-LSTR-AMC) peptidase activities in nuclear extracts. [1] - Addition of alloxan directly to normal NRK cell nuclear extract in vitro inhibited both peptidase activities in a dose-dependent manner, with maximal inhibition at approximately 3.3 mM. The inhibition was comparable to that of the proteasome-specific inhibitor PS-341. [1] - In a reconstituted GST-Sp1 degradation assay using NRK cell nuclear extract pretreated with 5 mM alloxan at 30°C for 30 min, alloxan inhibited the proteasome-dependent degradation of GST-Sp1 (conversion to GST-SpX fragments) as detected by anti-GST Western blot. [1] - Alloxan directly inhibited the chymotrypsin-like and trypsin-like peptidase activities of purified 26S proteasome in a dose-dependent manner, reaching maximum inhibition at about 3.3 mM. [1] - Alloxan directly inhibited the chymotrypsin-like and trypsin-like peptidase activities of purified 20S core particle in a dose-dependent manner, indicating that alloxan can enter the 20S cavity and directly act on the catalytic activity. [1] |
| ln Vivo |
Alloxan hydrate can be used in animal modeling to build rabbit and diabetic models.
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| Enzyme Assay |
Proteasome peptidase activity assay: Nuclear extract (10 µl) or purified 26S or 20S proteasome (0.1 µg) was mixed in 30 µl of proteasome activity assay buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 10% glycerol, 1 mM ATP, 1 mM DTT). Fluorogenic peptide substrates (100 µM suc-LLVY-AMC for chymotrypsin-like activity or 100 µM Boc-LSTR-AMC for trypsin-like activity) were added with or without alloxan. After incubation at 37°C for 90 minutes, reactions were stopped with 1% SDS (900 µl). Fluorescence intensity (excitation 360 nm, emission 450 nm) was measured using a fluorometer. To test the effect of alloxan in vitro, NRK cell nuclear extract was pretreated with alloxan at 30°C for 30 min before adding substrates. For dose-dependence, various concentrations of alloxan were tested, and maximal inhibition was observed at about 3.3 mM. [1]
- Sp1 degradation assay: GST-tagged Sp1 recombinant protein (10-20 ng) was added to NRK cell nuclear extract (1 µg/µl protein concentration) pretreated with or without 5 mM alloxan at 30°C for 30 min. The reaction was incubated at room temperature for 45 min, stopped with SDS sample buffer and boiling for 3 min. Samples were separated by SDS-PAGE, and GST-Sp1 and GST-SpX fragments were detected by anti-GST Western blot. [1] |
| Cell Assay |
NRK cell culture and treatment: NRK (normal rat kidney) cells were cultured in low glucose DMEM with 10% FBS, non-essential amino acids, and antibiotics. To test the effect of alloxan, 5 mM alloxan was added to the culture and incubated for 24 hr before harvesting. For detection of ubiquitinated proteins, cells were lysed with whole cell lysis buffer (50 mM Tris-HCl pH 8.0, 20% glycerol, 500 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 0.5% NP-40, 1 mM DTT, 1 mM PMSF). After centrifugation, pellets (containing insoluble ubiquitinated proteins) were dissolved in 1X SDS sample buffer (60 mM Tris-HCl pH 6.8, 120 mM DTT, 2.4% SDS, 6% glycerol, 0.12% bromophenol blue), sonicated, resolved by SDS-PAGE, and Western blotted with anti-ubiquitin antibody. [1]
- 293 cell transfection and GFP-CL1 degradation assay: 293 cells were cultured in DMEM with 10% FBS and antibiotics. 2.5 µg of pcDNA3.1-GFP-CL1 (or empty vector) was transfected using lipofectamine 3000 in 6-well plates without antibiotics. After 20 hr incubation, medium was changed to glucose-free medium containing 10 mM alloxan plus 50 µg/ml cycloheximide. Cells were harvested at indicated times and lysed with whole cell lysis buffer. Fluorescence intensity (excitation 460 nm, emission 535 nm) was measured and normalized to protein concentration. [1] - Nuclear extract preparation from NRK cells: Cells treated with or without alloxan were harvested and nuclear extract was prepared as described previously. The chymotrypsin-like and trypsin-like peptidase activities in the nuclear extract were measured using fluorogenic substrates as described under Enzyme Assay. [1] |
| ADME/Pharmacokinetics |
Stability: half-life of alloxan in phosphate buffer (50 mmol/l, pH 7.4) was 4.73 ± 0.12 min at 25°C and 1.38 ± 0.05 min at 37°C. [2]
Partition coefficient (log P value) in octanol/water at pH 2 was -1.84 ± 0.04, indicating high hydrophilicity. [2] |
| Toxicity/Toxicokinetics |
The toxicity of alloxan toward pancreatic β-cells is attributed to its thiol group reactivity. The central 5-carbonyl group reacts with thiol groups, potentially inhibiting thiol enzymes such as glucokinase. Inhibition of glucokinase reduces glucose oxidation and ATP production. [1]
- In the presence of intracellular thiols, alloxan generates reactive oxygen species (ROS) through a cyclic reaction with its reduction product dialuric acid, which is proposed to initiate its toxic action. [1] - The study shows that alloxan causes accumulation of ubiquitinated proteins in NRK cells and induces proteasome dysfunction, which may contribute to β-cell apoptosis.[1] |
| References | |
| Additional Infomation |
Alloxan and streptozotocin (STZ) are the two most prominent diabetogenic agents. Both are glucose analogues that preferentially accumulate in pancreatic β-cells via the low-affinity glucose transporter 2 (GLUT2), explaining their β-cell specificity. [1]
- Unlike STZ, which inhibits O-GlcNAcase and increases O-GlcNAcylation, alloxan inhibits O-GlcNAc transferase (OGT) and does not increase O-GlcNAcylation. However, both agents ultimately inhibit proteasome function: STZ indirectly via O-GlcNAcylation, and alloxan directly as a proteasome inhibitor. [1] - The study suggests that alloxan, being a uracil derivative containing multiple peptide bonds in its aromatic heterocyclic structure, may be recognized by the catalytic center of the proteasome, competing with substrates. [1] - Proteasome inhibition may play an important role in the toxic action of both alloxan and STZ toward pancreatic β-cells. The pathophysiological roles of proteasomes in β-cell failure and metabolic disorders deserve further exploration. [1] |
| Molecular Formula |
C4H4N2O5
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|---|---|
| Molecular Weight |
160.0850
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| Exact Mass |
160.012
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| CAS # |
2244-11-3
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| PubChem CID |
16723
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| Appearance |
White to yellow solid powder
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| Density |
1.681g/cm3
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| Boiling Point |
374.1ºC at 760 mmHg
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| Melting Point |
255 °C (dec.)(lit.)
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| Flash Point |
180ºC
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| Vapour Pressure |
8.59E-07mmHg at 25°C
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
11
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| Complexity |
222
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
DSXMTJRUNLATRP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C4H2N2O4.H2O/c7-1-2(8)5-4(10)6-3(1)9;/h(H2,5,6,8,9,10);1H2
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| Chemical Name |
1,3-diazinane-2,4,5,6-tetrone;hydrate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO : ~100 mg/mL (~624.69 mM)
H2O : ≥ 100 mg/mL (~624.69 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (15.62 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 25.0 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: ≥ 2.5 mg/mL (15.62 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 25.0 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: ≥ 2.5 mg/mL (15.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 6.2465 mL | 31.2324 mL | 62.4649 mL | |
| 5 mM | 1.2493 mL | 6.2465 mL | 12.4930 mL | |
| 10 mM | 0.6246 mL | 3.1232 mL | 6.2465 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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