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| Targets |
EN40 targets aldehyde dehydrogenase 3A1 (ALDH3A1), an enzyme that plays a role in the detoxification of aldehydes and is involved in cellular metabolism. ALDH3A1 is overexpressed in several cancers, including lung cancer, and is associated with poor prognosis and resistance to chemotherapy. By inhibiting ALDH3A1, EN40 disrupts cellular metabolism and redox balance, leading to impaired cancer cell survival. EN40 is a covalent inhibitor that targets a cysteine residue in the active site of ALDH3A1. This covalent binding leads to irreversible inhibition of the enzyme, which contributes to its potent and sustained activity. The compound has an IC50 of 2 μM for ALDH3A1. Its selectivity for ALDH3A1 over other aldehyde dehydrogenases is an important feature for minimizing off-target effects. By targeting ALDH3A1, EN40 provides a novel approach to cancer therapy, particularly for tumors that are dependent on this enzyme for survival and growth. The compound's ability to impair lung cancer pathogenicity both in situ and in vivo highlights its potential as a therapeutic agent for lung cancer.
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| ln Vitro |
EN40 demonstrates potent in vitro activity as an inhibitor of ALDH3A1 and as an anti-cancer agent. It inhibits ALDH3A1 with an IC50 of 2 μM. In cell-based assays, EN40 (10–1000 μM, 48 h) significantly inhibits ALDH3A1 activity and reduces A549 lung cancer cell survival. This demonstrates its ability to block the enzymatic activity of ALDH3A1 in a cellular context and to inhibit the growth of cancer cells. The compound's effects are concentration-dependent, with higher concentrations leading to greater inhibition of cell survival. EN40 is a covalent inhibitor, meaning it forms a stable bond with its target, leading to sustained inhibition. This covalent mechanism contributes to its potency and selectivity. The compound's potent activity against A549 lung cancer cells highlights its potential as a therapeutic agent for lung cancer.
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| ln Vivo |
EN40 (ip; 50 mg/kg; once daily beginning on day 14) has strong anti-tumorigenic effects in xenografts of existing A549 tumors and is well tolerated in mice that do not lose weight.
EN40 demonstrates significant in vivo activity, suppressing tumor growth in a mouse xenograft model. Intraperitoneal administration of EN40 (50 mg/kg, daily for 14 days) strongly suppresses tumor growth in established A549 xenografts. This demonstrates its efficacy in a relevant in vivo model of human lung cancer. Importantly, the treatment is well-tolerated, with no weight loss observed in treated mice, indicating a favorable safety profile. The compound's ability to impair lung cancer pathogenicity in vivo further supports its potential as a therapeutic agent. These in vivo results demonstrate that EN40 is a potent and effective ALDH3A1 inhibitor with significant anti-tumor activity. |
| Enzyme Assay |
The in vitro enzyme/receptor binding (non-cellular) assay for EN40 typically involves assessing its inhibitory activity against ALDH3A1 using a biochemical assay. In a typical protocol, purified ALDH3A1 enzyme is incubated with a substrate (e.g., aldehyde) and NAD+ in the presence of varying concentrations of EN40. The enzymatic activity is measured by monitoring the reduction of NAD+ to NADH spectrophotometrically. The inhibition of enzyme activity by EN40 is used to calculate the IC50. For EN40, this method has been used to determine its IC50 of 2 μM for ALDH3A1. To assess its selectivity, EN40 can be screened against a panel of other aldehyde dehydrogenases. These assays are crucial for characterizing the compound's inhibitory potency and selectivity at the molecular level.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: A549 cells Tested Concentrations: 10 μM; EN40 (10-1000 μM; 48 hrs (hours)) has an inhibitory effect on ALDH3A1 activity and reduces A549 cell death[1]. 100μM; 1000 μM Incubation Duration: 48 hrs (hours) Experimental Results: Inhibits A549 cell survival. The in vitro cellular assay for EN40 typically involves evaluating its effects on ALDH3A1 activity and cancer cell survival. In a typical assay, A549 lung cancer cells are treated with varying concentrations of EN40 (10–1000 μM) for 48 hours. After incubation, the cells are lysed, and ALDH3A1 activity is measured using a biochemical assay. Cell viability is also measured using an MTT or similar assay. The results show that EN40 significantly inhibits ALDH3A1 activity and reduces A549 cell survival in a concentration-dependent manner. These cellular assays are crucial for confirming the compound's mechanism of action and for evaluating its potency and efficacy in a relevant biological context. |
| Animal Protocol |
Animal/Disease Models: A549 cell SCID (severe combined immunodeficient) mouse [1]
Doses: 50 mg/kg Route of Administration: intraperitoneal (ip) injection; [1]. 50 mg/kg; from 14 days; one time/day Experimental Results: Strong anti-tumor effect on tumor xenografts. The in vivo animal experimental protocol for EN40 typically involves the use of a mouse xenograft model of lung cancer. In a typical study, immunodeficient mice are implanted with A549 human lung cancer cells subcutaneously. When tumors reach a certain size, the mice are treated with EN40, typically via intraperitoneal administration, at a dose of 50 mg/kg daily for 14 days. Tumor volume is measured regularly to assess tumor growth inhibition. Body weight is also monitored to assess tolerability. At the end of the study, tumors can be collected to analyze ALDH3A1 activity and markers of apoptosis or proliferation. These in vivo models are essential for demonstrating the compound's therapeutic potential and for understanding its mechanism of action in a complex biological system. |
| ADME/Pharmacokinetics |
Specific pharmacokinetic (PK) data for EN40, such as half-life, clearance, volume of distribution, or bioavailability, are not reported in the available literature. However, the compound's in vivo efficacy after intraperitoneal administration suggests that it reaches sufficient systemic concentrations to exert its pharmacological effects. EN40 has a molecular weight of 217.26 and is soluble in DMSO. The product is noted to be unstable in solution, suggesting that it should be prepared fresh before use. Further studies are needed to fully characterize the PK properties of EN40.
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| Toxicity/Toxicokinetics |
Specific toxicity (toxicology) data for EN40 are not reported in the available literature. However, the compound is well-tolerated in vivo at the tested dose (50 mg/kg, daily for 14 days), with no weight loss observed in treated mice. This suggests that it has a favorable safety profile at this dose. Comprehensive toxicological assessments, including acute and chronic toxicity studies, genotoxicity, and carcinogenicity, would be necessary to fully evaluate the safety profile of EN40 for clinical development. The compound is for research use only.
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| References | |
| Additional Infomation |
EN40 is a novel, potent, and selective aldehyde dehydrogenase 3A1 (ALDH3A1) inhibitor that functions as a covalent ligand. It has an IC50 of 2 μM for ALDH3A1. EN40 is a cysteine-targeted covalent inhibitor. In vitro, it inhibits ALDH3A1 activity and reduces A549 lung cancer cell survival. In vivo, it suppresses tumor growth in A549 xenografts. The compound has a molecular formula of C13H15NO2 and a molecular weight of 217.26. EN40 represents a valuable chemical probe and therapeutic candidate for studying ALDH3A1 biology and tumor metabolism.
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| Molecular Formula |
C13H15NO2
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| Molecular Weight |
217.263703584671
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| Exact Mass |
217.11
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| CAS # |
2094547-67-6
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| PubChem CID |
126824761
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| Appearance |
Light yellow to yellow ointment
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| LogP |
1.2
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
16
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| Complexity |
333
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=C(CN(CC1)C(=O)C=C)C2=CC=CO2
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| InChi Key |
UYNKILJNUUDFFJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H15NO2/c1-3-13(15)14-7-6-10(2)11(9-14)12-5-4-8-16-12/h3-5,8H,1,6-7,9H2,2H3
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| Chemical Name |
1-[5-(furan-2-yl)-4-methyl-3,6-dihydro-2H-pyridin-1-yl]prop-2-en-1-one
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| Synonyms |
EN40 EN 40 EN-40
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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). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. (2). This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| 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 (~460.28 mM)
Ethanol : ~100 mg/mL (~460.28 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 | 4.6028 mL | 23.0139 mL | 46.0278 mL | |
| 5 mM | 0.9206 mL | 4.6028 mL | 9.2056 mL | |
| 10 mM | 0.4603 mL | 2.3014 mL | 4.6028 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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