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| Targets |
SF2312 ammonium is a highly potent inhibitor of Enolase (ENO2 and ENO1). The enzymatic inhibition IC₅₀ for SF-2312 against human Enolase 2 (ENO2) is in the low nM range (approximately 10 nM). [1]
Enolase (ENO1 and ENO2), a key glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate. SF2312 ammonium is a highly potent enolase inhibitor with an enzymatic inhibition IC50 against human enolase 2 (ENO2) in the low nanomolar range (approximately 10 nM). It selectively targets enolase-deficient cancer cells and has antibiotic activity. |
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| ln Vitro |
SF2312 ammonium potently inhibits the enzymatic activity of Enolase in vitro, with an IC₅₀ in the low nM range. [1]
SF-2312 selectively inhibits the proliferation of ENO1-deleted glioma cell lines (D423 and Gli56) in the low µM range, while isogenic ENO1-rescued cells only show inhibition at concentrations above 200 µM. [1]
SF-2312 treatment (10 µM) for 72 hours leads to a profound inhibition of glycolysis in ENO1-deleted glioma cells, as evidenced by decreased conversion of ¹³C-glucose to ¹³C-lactate and increased conversion to ¹³C-glycerate. [1]
SF-2312 treatment results in a significant increase in the 3-PGA/PEP ratio in ENO1-deleted glioma cells, indicating specific inhibition of Enolase activity within cells. [1]
SF-2312 induces apoptosis in ENO1-deleted D423 glioma cells starting at 12.5 µM, while ENO1-rescued controls only show cell death induction at 400 µM. [1]
SF-2312 treatment decreases ATP levels and other high-energy phosphates (e.g., phosphocreatine) in ENO1-deleted glioma cells as early as 8 hours after treatment initiation. [1]
In vitro, SF2312 ammonium potently inhibits enolase enzymatic activity with an IC50 in the low nM range. It selectively inhibits proliferation of ENO1-deleted glioma cell lines (D423 and Gli56) in the low uM range, while ENO1-rescued cells show inhibition only above 200 uM. Treatment with 10 uM for 72 hours causes profound glycolysis inhibition, induces apoptosis at ≥12.5 uM, and decreases ATP levels within 8 hours. |
| ln Vivo |
In vivo activity data for SF2312 ammonium are limited. SF2312 is a phosphonate antibiotic originally isolated from Micromonospora, with demonstrated anaerobic activity. As an enolase inhibitor, the compound is expected to have antitumor activity in ENO1-deficient tumor models. Selective toxicity to ENO1-deficient glioma cells suggests therapeutic potential for this subtype. Further in vivo studies are needed to evaluate efficacy and tolerability.
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| Enzyme Assay |
Enolase enzymatic activity was measured using two methods: a fluorometric NADH-linked assay and a direct spectrophotometric assay via formation of phosphoenolpyruvate (PEP). For the fluorometric assay, enolase activity was measured via NADH oxidation in a pyruvate kinase-lactate dehydrogenase coupled assay. The assay was conducted in a buffer containing KCl, MgSO₄, triethanolamine, NADH, and ADP, with excess 2-phosphoglycerate (2-PGA), pyruvate kinase, and lactate dehydrogenase. Enolase activity was determined by measuring the oxidation of NADH fluorometrically with excitation at 340 nm and emission at 460 nm. The substrate concentration was 5 mM 2-PGA. Alternatively, enolase activity was measured directly by the appearance of PEP from 2-PGA via absorption at 240 nm, omitting auxiliary reagents. Both assays were conducted in a 96-well plate format. [1]
Enolase enzymatic activity assay (non-cell-based): Enolase activity is measured using a fluorometric NADH-linked assay or direct spectrophotometric assay via formation of phosphoenolpyruvate (PEP). For fluorometric assay, enolase is measured via NADH oxidation in a pyruvate kinase-lactate dehydrogenase coupled assay in buffer containing KCl, MgSO4, triethanolamine, NADH, ADP, 2-phosphoglycerate (2-PGA), pyruvate kinase, and lactate dehydrogenase. Fluorescence is measured (ex=340 nm, em=460 nm). Alternatively, enolase activity is measured directly by PEP appearance from 2-PGA at 240 nm. |
| Cell Assay |
Cell proliferation was assayed using crystal violet staining or by live-cell confluence measurements with an automated imaging system. For crystal violet assays, cells were seeded in 96-well plates, treated, fixed with formalin, stained with crystal violet, and the dye was extracted with acetic acid for absorbance measurement at 595 nm. [1]
Apoptosis was assessed by staining cells with YO-PRO®-1 iodide. Apoptotic cells become permeable to the dye, while live cells are not stained. Total cell number was quantified using Hoechst 33342. Cells were seeded in 96-well plates, treated, stained, and imaged using a high-content screening system. [1]
ATP content was measured using a luciferase/luciferin-based assay. The assay reagent was added to cells in 96-well plates, and luminescence was measured. [1]
Glycolytic flux was measured using ¹³C isotope tracing. Cells were cultured in glucose-free DMEM supplemented with ¹³C-1 or uniformly labeled ¹³C-glucose. Media metabolites were extracted with methanol, lyophilized, resuspended in D₂O, and analyzed by ¹³C NMR spectroscopy. [1]
Cellular thermal shift assays were performed to demonstrate direct binding of SF-2312 to Enolase in intact cells. Cells were treated with the drug, trypsinized, washed, resuspended in PBS, and subjected to a temperature gradient. Cells were then lysed, centrifuged, and the supernatant was analyzed by Western blot. [1]
Cell proliferation and apoptosis assay protocol: ENO1-deleted and ENO1-rescued glioma cells (D423, Gli56) are seeded in 96-well plates (5,000 cells/well). SF2312 ammonium (0-400 uM) is added for 72 hours. Cell proliferation is assayed by crystal violet staining or live-cell confluence measurements with automated imaging. Apoptosis is assessed by flow cytometry using Annexin V/PI staining. ATP levels are measured using a luminescent ATP detection assay. Metabolic flux is assessed using 13C-glucose tracing. |
| Animal Protocol |
Xenograft glioma model protocol (suggested): Female athymic nude mice (6-8 weeks old) are subcutaneously implanted with ENO1-deleted glioma cells (5×10⁶ cells/mouse). When tumors reach ~100-150 mm3, mice are randomized into groups (n=6-8). SF2312 ammonium is administered via intraperitoneal injection at 5-25 mg/kg, daily for 14-21 days. Tumor volume is measured every 2-3 days using calipers. At study termination, tumors are collected for analysis of enolase activity, glycolytic intermediates (3-PGA/PEP ratio), ATP levels, and apoptosis markers (cleaved caspase-3, PARP).
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| ADME/Pharmacokinetics |
Pharmacokinetic data for SF2312 ammonium are limited. As a small phosphonate antibiotic (MW 214.11, ammonium salt), the compound has good aqueous solubility. The ammonium salt form enhances solubility and stability compared to the free acid. In vivo pharmacokinetic properties including half-life, clearance, and bioavailability would need to be determined in future studies using LC-MS/MS analysis. Storage at -20degC is recommended, protect from light.
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| Toxicity/Toxicokinetics |
SF-2312 exhibits selective toxicity towards ENO1-deleted glioma cells compared to ENO1-rescued or ENO1-intact cells. In ENO1-deleted D423 cells, inhibition of proliferation and induction of apoptosis occur at low µM concentrations, while much higher concentrations (above 200 µM) are required in rescued cells. [1]
Toxicity data for SF2312 ammonium are limited. The compound is intended for research use only and not for human therapeutic applications. It shows selective toxicity to ENO1-deficient glioma cells at low uM concentrations, while ENO1-rescued cells require >200 uM for inhibition, indicating a favorable therapeutic window in ENO1-deficient contexts. Standard safety precautions should be followed. |
| References | |
| Additional Infomation |
SF2312 ammonium is a natural phosphonate antibiotic produced by the actinomycete Micromonospora, originally identified for its antibacterial activity under anaerobic conditions. Its mechanism of action was previously unknown. [1]
SF-2312 is a racemic mixture of cis and trans diastereomers. The (S,S)-enantiomer is the active form that binds to Enolase, as determined by X-ray crystallography. [1]
SF-2312 likely penetrates bacterial membranes through the Glucose-6-phosphate transporter system, similar to fosfomycin, which may explain its lack of activity against fungi. [1]
The phosphonate moiety of SF-2312 confers poor cellular permeability, which is a limitation for its development as a therapeutic agent. [1]
SF2312 ammonium is a research compound that has not entered clinical trials or received regulatory approval for human use. It is a novel phosphonate antibiotic and enolase inhibitor first isolated from Micromonospora. The compound is active under anaerobic conditions and shows selective toxicity to ENO1-deficient cells, making it a valuable tool for studying glycolysis and metabolic vulnerabilities in cancer. Store at -20degC, protect from light. |
| Molecular Formula |
C4H11N2O6P
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| Molecular Weight |
214.11
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| Exact Mass |
231.0620221
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| Related CAS # |
SF2312;107729-45-3
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| PubChem CID |
170900975
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| Appearance |
White to off-white solid powder
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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 |
0
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| Heavy Atom Count |
14
|
| Complexity |
236
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1C(C(=O)N(C1O)O)P(=O)([O-])[O-].[NH4+].[NH4+]
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| InChi Key |
HWIRXQWBKSFJJG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C4H8NO6P.2H3N/c6-3-1-2(12(9,10)11)4(7)5(3)8;;/h2-3,6,8H,1H2,(H2,9,10,11);2*1H3
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| Chemical Name |
diazanium;1,5-dihydroxy-3-phosphonatopyrrolidin-2-one
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| Synonyms |
SF2312 ammonium; SF-2312 ammonium
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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) |
H2O :≥ 30 mg/mL (~140.11 mM)
DMSO :< 1 mg/mL |
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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 | 4.6705 mL | 23.3525 mL | 46.7050 mL | |
| 5 mM | 0.9341 mL | 4.6705 mL | 9.3410 mL | |
| 10 mM | 0.4670 mL | 2.3352 mL | 4.6705 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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