| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Transketolase (TKT). Benfooxythiamine (BOT) is a prodrug of oxythiamine and acts as an irreversible inhibitor of transketolase (TKT), a key enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP). [1]
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|---|---|
| ln Vitro |
BOT inhibited SARS-CoV-2 replication in Caco-2 cells (human colon adenocarcinoma cell line) infected with two different SARS-CoV-2 isolates (FFM1 and FFM7) in a concentration-dependent manner, as indicated by reduced viral Spike (S) protein levels. Non-toxic concentrations of BOT were used. [1]
In air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells infected with SARS-CoV-2 FFM7 (MOI 1), BOT inhibited viral replication as quantified by viral genomic RNA copy numbers. [1] BOT increased the anti-SARS-CoV-2 activity of the glycolysis inhibitor 2-deoxy-D-glucose (2DG). The combination of BOT and 2DG resulted in greater reduction of cellular S protein levels and viral genomic RNA copy numbers compared to BOT alone. [1] |
| Enzyme Assay |
BOT inhibited SARS-CoV-2 replication in Caco-2 cells (human colon adenocarcinoma cell line) infected with two different SARS-CoV-2 isolates (FFM1 and FFM7) in a concentration-dependent manner, as indicated by reduced viral Spike (S) protein levels. Non-toxic concentrations of BOT were used. [1]
In air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells infected with SARS-CoV-2 FFM7 (MOI 1), BOT inhibited viral replication as quantified by viral genomic RNA copy numbers. [1] BOT increased the anti-SARS-CoV-2 activity of the glycolysis inhibitor 2-deoxy-D-glucose (2DG). The combination of BOT and 2DG resulted in greater reduction of cellular S protein levels and viral genomic RNA copy numbers compared to BOT alone. [1] |
| Cell Assay |
Antiviral and Cytotoxicity Assay (Caco-2 cells): Caco-2 cells were seeded in 96-well plates. After reaching confluency, cells were pre-treated with BOT for 24 hours, then infected with SARS-CoV-2 at MOI 0.01. Antiviral effects were determined by immunostaining for SARS-CoV-2 Spike protein and/or quantification of viral genomes by qRT-PCR. Cytotoxic effects were determined by MTT assay. IC₅₀ and CC₅₀ values were determined using curve regression. [1]
Immunostaining: To detect SARS-CoV-2 Spike protein, infected cells were fixed with acetone:methanol (40:60) solution, incubated with a primary monoclonal antibody against SARS-CoV-2 Spike (1:1500), followed by a peroxidase-conjugated anti-rabbit secondary antibody (1:1000) and AEC substrate. Staining was quantified using a BIOREADER-7000-F-z-I. [1] qRT-PCR for Viral Genome: RNA from cell culture supernatant was isolated using a viral RNA kit. Viral RNA was detected using primers targeting the RNA-dependent RNA polymerase (RdRp) gene. A standard curve generated by plasmid DNA containing the RdRp target sequence was used to determine viral copy numbers. [1] Primary Human Bronchial Epithelial (HBE) Air-Liquid Interface (ALI) Culture: HBE cells were differentiated into ALI cultures. Cells were infected with SARS-CoV-2 FFM7 at MOI 1 from the apical side. After 2 hours, the inoculum was removed, cells were washed, and BOT was added from both the apical and basal sides. Apical treatment was removed after one day. Genomic viral RNA copy numbers were determined after five days. Cytotoxicity was determined by LDH-Glo™ Cytotoxicity Assay. [1] Combination Treatment with 2DG: Caco-2 cells were pre-treated with different concentrations of BOT for 24 hours, then 2DG (5 mM or 10 mM) was added and cells were infected with SARS-CoV-2 FFM7 at MOI 0.01. Antiviral effects were assessed by immunostaining and qRT-PCR as described above. [1] |
| Toxicity/Toxicokinetics |
Antiviral and Cytotoxicity Assay (Caco-2 cells): Caco-2 cells were seeded in 96-well plates. After reaching confluency, cells were pre-treated with BOT for 24 hours, then infected with SARS-CoV-2 at MOI 0.01. Antiviral effects were determined by immunostaining for SARS-CoV-2 Spike protein and/or quantification of viral genomes by qRT-PCR. Cytotoxic effects were determined by MTT assay. IC₅₀ and CC₅₀ values were determined using curve regression. [1]
Immunostaining: To detect SARS-CoV-2 Spike protein, infected cells were fixed with acetone:methanol (40:60) solution, incubated with a primary monoclonal antibody against SARS-CoV-2 Spike (1:1500), followed by a peroxidase-conjugated anti-rabbit secondary antibody (1:1000) and AEC substrate. Staining was quantified using a BIOREADER-7000-F-z-I. [1] qRT-PCR for Viral Genome: RNA from cell culture supernatant was isolated using a viral RNA kit. Viral RNA was detected using primers targeting the RNA-dependent RNA polymerase (RdRp) gene. A standard curve generated by plasmid DNA containing the RdRp target sequence was used to determine viral copy numbers. [1] Primary Human Bronchial Epithelial (HBE) Air-Liquid Interface (ALI) Culture: HBE cells were differentiated into ALI cultures. Cells were infected with SARS-CoV-2 FFM7 at MOI 1 from the apical side. After 2 hours, the inoculum was removed, cells were washed, and BOT was added from both the apical and basal sides. Apical treatment was removed after one day. Genomic viral RNA copy numbers were determined after five days. Cytotoxicity was determined by LDH-Glo™ Cytotoxicity Assay. [1] Combination Treatment with 2DG: Caco-2 cells were pre-treated with different concentrations of BOT for 24 hours, then 2DG (5 mM or 10 mM) was added and cells were infected with SARS-CoV-2 FFM7 at MOI 0.01. Antiviral effects were assessed by immunostaining and qRT-PCR as described above. [1] |
| References | |
| Additional Infomation |
Benfooxythiamine (BOT) is an oxythiamine prodrug that irreversibly inhibits transketolase (TKT), a key enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP). [1]
SARS-CoV-2 infection was associated with increased transketolase (TKT) levels in infected cells based on proteomics data, suggesting a role for the non-oxidative PPP in viral replication. [1] BOT inhibits SARS-CoV-2 replication and increases the anti-SARS-CoV-2 activity of the glycolysis inhibitor 2-deoxy-D-glucose (2DG), which is under development for COVID-19 therapy. The combination targets ribose-5-phosphate production: 2DG reduces fructose-6-phosphate and glucose-6-phosphate (precursors for ribose-5-phosphate), while BOT directly inhibits TKT in the non-oxidative PPP. [1] Metabolic drugs like BOT and 2DG may also interfere with COVID-19-associated immunopathology by modifying the metabolism of immune cells in addition to inhibiting viral replication. [1] |
| Molecular Formula |
C19H22N3O7PS
|
|---|---|
| Molecular Weight |
467.432644367218
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| CAS # |
909542-99-0
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| Appearance |
Typically exists as solids at room temperature
|
| LogP |
2.977
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| SMILES |
C/C(=C(\SC(C1C=CC=CC=1)=O)/CCOP(=O)(O)O)/N(C=O)CC1C(=O)NC(C)=NC=1
|
| Chemical Name |
（E)-S-(2-(N-((2-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)methyl)formamido)-5-(phosphonooxy)pent-2-en-3-yl) benzothioate
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1394 mL | 10.6968 mL | 21.3936 mL | |
| 5 mM | 0.4279 mL | 2.1394 mL | 4.2787 mL | |
| 10 mM | 0.2139 mL | 1.0697 mL | 2.1394 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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