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| 5mg |
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| 10mg |
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| 25mg |
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| Targets |
EN460 selectively targets the endoplasmic reticulum oxidation 1 (ERO1) enzyme, specifically binding to the reduced active form of ERO1α and preventing its re-oxidation. By inhibiting ERO1, EN460 disrupts the oxidative protein folding pathway in the endoplasmic reticulum. This inhibition has been shown to protect cells from erastin-induced ferroptosis and to inhibit cell migration and invasion in pancreatic ductal adenocarcinoma (PDAC) models.
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| ln Vitro |
Reducing cells exposed to EN460 or QM295 accumulate less mobile ERO1α forms, which represent the cell's state. In cultivated 293T cells, it was also discovered that QM295 and EN460 activated the unfolded protein response reporter gene [1].
In vitro, EN460 is a selective inhibitor of ERO1α with an IC₅₀ of 1.9 µM. It selectively interacts with the reduced active form of ERO1α and prevents its re-oxidation. By inhibiting ERO1L, EN460 has been shown to inhibit cell migration and invasion in PDAC models. It also protects cells from erastin-induced ferroptosis. In zebrafish embryos, EN460 showed a dose-dependent effect, with a dose of 10 µM being significantly lethal during early embryonic development. |
| ln Vivo |
In vivo activity for EN460 has not been extensively documented in the available literature. Its role as a selective ERO1 inhibitor suggests potential use in animal models to study the role of ERO1 in cancer progression and metastasis. The compound's ability to protect cells from ferroptosis could also be explored in models of ferroptosis-related diseases.
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| Enzyme Assay |
Non-cellular enzyme assays for EN460 are based on its inhibition of ERO1α activity. These assays typically use a reduced form of ERO1α and a fluorescent substrate, such as a dihydrofluorescein-based probe, to measure the enzyme's re-oxidation activity. The enzyme is incubated with the substrate and varying concentrations of EN460. The increase in fluorescence, which corresponds to the oxidation of the substrate, is monitored over time. The IC₅₀ value for inhibition is determined from the dose-response curves.
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| Cell Assay |
In vitro cellular assays for EN460 involve treating cells, such as PDAC cell lines, with the compound to assess its effects on cell migration, invasion, and ferroptosis. To evaluate migration and invasion, cells are treated with EN460 and then subjected to wound healing or transwell invasion assays. The number of cells that migrate or invade through the membrane is quantified. To study ferroptosis, cells are treated with erastin to induce ferroptosis, and the protective effect of EN460 is measured by assessing cell viability using MTT or CellTiter-Glo® assays.
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| Animal Protocol |
In vivo animal experiments for EN460 are not extensively documented in the available literature. Given its mechanism as an ERO1 inhibitor, potential studies could involve administering EN460 to mouse xenograft models of PDAC to evaluate its effects on tumor growth, migration, and invasion. Endpoints would include tumor volume measurements, assessment of metastasis, and evaluation of pharmacodynamic markers such as ERO1 activity and markers of oxidative stress in tumor tissue.
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| ADME/Pharmacokinetics |
EN460 has a molecular weight of 460.79 and a molecular formula of C₂₂H₁₂ClF₃N₂O₄. It is a solid powder with a purity of 99.67%. The compound is soluble in DMSO (17 mg/mL) but insoluble in water and ethanol. For storage, the powder should be kept at -20°C for up to 3 years or at 4°C for up to 2 years; stock solutions in solvent can be stored at -80°C for 6 months or at -20°C for 1 month.
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| Toxicity/Toxicokinetics |
EN460 is a research compound for laboratory use only and is not intended for human therapeutic or diagnostic use. In zebrafish embryos, a dose of 10 µM was found to be significantly lethal during early embryonic development. Standard laboratory safety precautions should be followed when handling the compound. It may cause skin, eye, and respiratory irritation. Appropriate personal protective equipment should be used.
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| References | |
| Additional Infomation |
2-chloro-5-[(4Z)-5-oxo-4-[(5-phenylfuran-2-yl)methylidene]-3-(trifluoromethyl)pyrazol-1-yl]benzoic acid is an organic molecular entity that has inhibitory effects.
EN460 (CAS 496807-64-8) is a selective small-molecule inhibitor of endoplasmic reticulum oxidation 1 (ERO1) with an IC₅₀ of 1.9 µM. It specifically targets the reduced active form of ERO1α and is a valuable tool for studying the role of ERO1 in cancer cell migration, invasion, and ferroptosis. It is available from various commercial suppliers for research applications. |
| Molecular Formula |
C22H12CLF3N2O4
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|---|---|
| Molecular Weight |
460.7932
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| Exact Mass |
460.043
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| CAS # |
496807-64-8
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| PubChem CID |
1016023
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| Appearance |
Pink to red solid powder
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| LogP |
3.285
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
810
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C(C=C1)C2=CC=C(O2)/C=C\3/C(=NN(C3=O)C4=CC(=C(C=C4)Cl)C(=O)O)C(F)(F)F
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| InChi Key |
RSLFQCNAOMQAIH-WJDWOHSUSA-N
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| InChi Code |
InChI=1S/C22H12ClF3N2O4/c23-17-8-6-13(10-15(17)21(30)31)28-20(29)16(19(27-28)22(24,25)26)11-14-7-9-18(32-14)12-4-2-1-3-5-12/h1-11H,(H,30,31)/b16-11-
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| Chemical Name |
2-chloro-5-[(4Z)-5-oxo-4-[(5-phenylfuran-2-yl)methylidene]-3-(trifluoromethyl)pyrazol-1-yl]benzoic acid
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| Synonyms |
EN-460EN460 EN 460.
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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 : ~12.5 mg/mL (~27.13 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: ≥ 1.25 mg/mL (2.71 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 12.5 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. View More
Solubility in Formulation 3: 1.25 mg/mL (2.71 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1702 mL | 10.8509 mL | 21.7019 mL | |
| 5 mM | 0.4340 mL | 2.1702 mL | 4.3404 mL | |
| 10 mM | 0.2170 mL | 1.0851 mL | 2.1702 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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