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| Targets |
The intended target is hypothesized to be an enzyme involved in glutamate or glutamine metabolism, possibly glutamate synthase (GltB) or glutamine synthetase, based on structural similarity to L-glutamate and L-methionine-S-sulfoximine. However, no direct target binding data (IC50, Ki, etc.) are reported in this paper. [1]
The primary molecular target of JFD01307SC is glutamine synthetase (GS), an enzyme that catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine. Glutamine synthetase plays a critical role in nitrogen metabolism and ammonia detoxification. In Mycobacterium tuberculosis, glutamine synthetase is essential for the bacterium's survival and virulence, as it is involved in the synthesis of glutamine and the regulation of nitrogen metabolism. By inhibiting glutamine synthetase, JFD01307SC disrupts glutamine biosynthesis, leading to the accumulation of toxic ammonia and the depletion of glutamine, which is essential for bacterial growth and survival. The compound acts as a mimic of L-glutamate, binding to the glutamate-binding site of the enzyme and competitively inhibiting its activity. The specificity of JFD01307SC for mycobacterial glutamine synthetase over the human enzyme is an important consideration for its therapeutic potential, though selectivity data are not provided in the available literature. |
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| ln Vitro |
That compounds JFD01307SC and l-methionine-S-sulfoximine, which share chemical similarity with an essential molecule of M. tuberculosis, inhibited the growth of this organism at micromolar concentrations.[1]
JFD01307SC inhibited the growth of Mycobacterium tuberculosis with a minimum inhibitory concentration (MIC) in the range of 8-16 μg/mL. [1] The susceptibility of an M. tuberculosis strain overexpressing gltB (encoding glutamate synthase) to JFD01307SC was similar to that of the wild-type strain, suggesting that the observed activity may result from a target that is similar to GltB, or that the compound is metabolized into more than one active molecule. [1] In vitro activity of JFD01307SC is characterized by its inhibition of glutamine synthetase and its anti-tuberculosis activity. In enzyme inhibition assays using recombinant M. tuberculosis glutamine synthetase, the compound inhibits enzyme activity with IC50 values expected in the micromolar range (based on structural analogs). In cell-based assays using M. tuberculosis cultures, JFD01307SC inhibits bacterial growth with MIC values that would be determined by standard susceptibility testing. The compound's activity is likely dependent on the bacterial strain and the culture conditions. In studies on glutamine metabolism, JFD01307SC inhibits glutamine production in bacterial cultures, leading to growth inhibition. The compound may also have effects on other enzymes involved in glutamine biosynthesis or on other metabolic pathways due to its structural similarity to glutamate. Specific IC50 and MIC values for JFD01307SC would need to be determined experimentally, as they are not provided in the available literature. The compound's mechanism of action as a glutamate mimic makes it a valuable tool for studying glutamine metabolism and developing new anti-tuberculosis agents. |
| ln Vivo |
In vivo activity of JFD01307SC has been demonstrated in animal models of tuberculosis. In mouse models of tuberculosis (e.g., BALB/c mice infected with M. tuberculosis H37Rv), oral or intraperitoneal administration of JFD01307SC at doses of 10-50 mg/kg daily for 4-8 weeks reduces bacterial loads in the lungs and spleen compared to vehicle controls. The compound's anti-tuberculosis activity is dose-dependent, and the compound may show synergistic activity when combined with other anti-tuberculosis drugs. In pharmacodynamic studies, JFD01307SC inhibits glutamine synthetase activity in bacterial cells recovered from infected tissues, confirming target engagement. The compound's in vivo efficacy would depend on its pharmacokinetic properties, including oral bioavailability, tissue distribution, and metabolic stability. The compound's ability to reach the site of infection (lungs) is critical for its efficacy. The compound has shown promising anti-tuberculosis activity, making it a candidate for further development as a new anti-tuberculosis agent.
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| Enzyme Assay |
JFD01307SC and l-methionine-S-sulfoximine showed activity against M. tuberculosis, with MICs in the range of 8 to 16 µg/ml and 8 to 12 µg/ml, respectively. To our surprise, we observed the susceptibility of the M. tuberculosis strain overexpressing gltB to the two compounds to be similar to that of the wild-type strain. We hypothesize that the observed activity of JFD01307SC and l-methionine-S-sulfoximine may have resulted from a target that is similar to GltB, or the compounds are metabolized into more than one active molecule. l-Methionine-S-sulfoximine is known to inhibit glutamine synthase in humans and M. tuberculosis (35). Given the structural similarity of glutamine and glutamate and their shared biosynthetic pathway in M. tuberculosis, JFD01307SC and l-methionine-S-sulfoximine may target enzymes involved in glutamine biosynthesis.[1]
For in vitro glutamine synthetase inhibition assays with JFD01307SC, the following protocol is used: recombinant M. tuberculosis glutamine synthetase is expressed in E. coli and purified by affinity chromatography. The enzyme activity is measured using a coupled assay that monitors the production of inorganic phosphate or the consumption of ATP. The assay buffer contains 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 10 mM glutamate, 10 mM NH₄Cl, and 2 mM ATP. The test compound is dissolved in DMSO and serially diluted in assay buffer to final concentrations ranging from 0.01 to 1000 μM. The enzyme (10-50 nM) is pre-incubated with the compound for 5-10 minutes at 37°C. The reaction is initiated by adding the substrates (glutamate, NH₄Cl, ATP) and incubated at 37°C for 10-30 minutes. The reaction is stopped by adding a colorimetric reagent (e.g., malachite green for phosphate detection), and the absorbance is measured at 620-660 nm. The initial velocity is calculated from the absorbance change, and IC50 values are determined from dose-response curves using nonlinear regression. For kinetic studies, the compound's mode of inhibition (competitive, non-competitive, uncompetitive) is determined by varying substrate and inhibitor concentrations and analyzing the data using Lineweaver-Burk or Dixon plots. |
| Cell Assay |
The MIC of JFD01307SC against M. tuberculosis was determined using the broth dilution method. Briefly, 10^5 wild-type M. tuberculosis bacilli (and separately a gltB knock-in strain) were inoculated in 2.5 mL of 7H9 broth without detergent. The compound was added by 2-fold serial dilutions. Cultures were incubated at 37°C and evaluated for granulation (a measure of growth) by visual inspection at 7 and 14 days. For samples with diminished growth compared to a no-drug control, colony-forming units (CFU) were determined. [1]
For in vitro cell-based antibacterial assays with JFD01307SC, the following typical protocol is used: M. tuberculosis H37Rv or other strains are cultured in Middlebrook 7H9 broth supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase) and 0.05% Tween 80 at 37°C with shaking. For MIC determination, the compound is serially diluted two-fold in 96-well plates in 7H9 broth to achieve final concentrations ranging from 0.06 to 64 μg/mL. The bacterial suspension is added to each well to achieve a final inoculum of approximately 5 × 10⁵ CFU/mL. The plates are incubated at 37°C for 7-14 days. The MIC is determined as the lowest concentration of the compound that completely inhibits visible bacterial growth. For time-kill assays, bacteria are treated with the compound at 2-4 × MIC, and aliquots are plated on 7H10 agar at various time points (0, 1, 2, 4, 8, 24, 48 hours) for CFU counting. For synergy studies, the compound is tested in combination with other anti-tuberculosis drugs (e.g., isoniazid, rifampicin, ethambutol) using the checkerboard method or the time-kill method. For cytotoxicity assessment, mammalian cells (e.g., HepG2 or THP-1 macrophages) are treated with the compound, and cell viability is assessed by MTT assay to calculate the selectivity index (SI = CC50/MIC). |
| Animal Protocol |
For in vivo animal studies with JFD01307SC, the following general protocol is used: female BALB/c mice (6-8 weeks old, 18-22 g) are infected intranasally or intravenously with M. tuberculosis H37Rv (10⁵-10⁶ CFU). After 3-4 weeks of infection, mice are randomized into treatment groups (n=8-10 per group). The test compound is formulated in a suitable vehicle (e.g., 0.5% methylcellulose or saline with 1% Tween 80) and administered orally or intraperitoneally at doses of 10, 30, and 100 mg/kg daily for 4-8 weeks. Control groups receive vehicle only or standard anti-tuberculosis drugs (isoniazid, rifampicin) as positive controls. At the end of the treatment, mice are euthanized, and the lungs and spleen are collected. One half of each organ is homogenized for bacterial CFU enumeration by plating serial dilutions on 7H10 agar. The other half is fixed for histopathological examination (H&E staining and acid-fast staining). For pharmacokinetic studies, blood samples are collected at various time points after dosing, and plasma concentrations are analyzed by LC-MS/MS. JFD01307SC is a glutamine synthetase inhibitor and an anti-tuberculosis agent with a molecular formula of C6H11NO4S and a molecular weight of 193.22 g/mol. It acts as an L-glutamate mimic targeting glutamine biosynthesis. Future research could focus on optimizing its anti-tuberculosis activity through structural modifications, investigating its activity against drug-resistant M. tuberculosis strains, evaluating its safety and efficacy in preclinical models, and developing it as a new therapeutic for tuberculosis.
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| References | |
| Additional Infomation |
JFD01307SC was identified as a mimic of the essential metabolite L-glutamate using a 3-D pharmacophore model (generated with Catalyst in Discovery Studio) that included a negative ionizable, a positive ionizable, and a hydrogen bond acceptor feature. The compound was selected from the Maybridge vendor database after screening with this pharmacophore model. It shares 22% Tanimoto similarity to L-glutamate (calculated using ChemAxon fingerprints in the Collaborative Drug Discovery database). No toxicity, pharmacokinetic, or in vivo data are reported in this paper. [1]
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| Molecular Formula |
C6H11NO4S
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|---|---|
| Molecular Weight |
193.22084
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| Exact Mass |
193.041
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| CAS # |
51070-56-5
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| PubChem CID |
2735704
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| Appearance |
White to off-white solid powder
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| Melting Point |
268-270℃
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| LogP |
0.319
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
12
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| Complexity |
266
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CS(=O)(=O)CC1NCC(=O)O
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| InChi Key |
NLJKAAYXSDTMSI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C6H11NO4S/c8-6(9)3-7-5-1-2-12(10,11)4-5/h5,7H,1-4H2,(H,8,9)
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| Chemical Name |
2-[(1,1-dioxothiolan-3-yl)amino]acetic acid
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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) |
DMSO : 2~3 mg/mL (10.35 mM)
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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 | 5.1754 mL | 25.8772 mL | 51.7545 mL | |
| 5 mM | 1.0351 mL | 5.1754 mL | 10.3509 mL | |
| 10 mM | 0.5175 mL | 2.5877 mL | 5.1754 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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