| Size | Price | Stock | Qty |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| Other Sizes |
| Targets |
MYC
Target: EN4 covalently targets the intrinsically disordered cysteine residue Cys275 within the MYC oncoprotein[1] |
|---|---|
| ln Vitro |
En4 inhibits MYC transcriptional activity, downregulates numerous MYC transcriptional targets, directly targets MYC in cells, decreases MYC and MAX thermal stability, and hinders tumorigenesis. Treatment with EN4 significantly reduces the proliferation of 231MFP breast cancer cells in a dose- and time-dependent manner. Mammary epithelial MCF10A cells that have undergone MYC transformation are less likely to survive when exposed to EN4, but not parental MCF10A cells that are not MYC-dependent. [1]
Target Binding Activity: EN4 covalently binds to the disordered Cys275 site of MYC in biochemical assays, with target engagement verified by mass spectrometry and biophysical methods, and it disrupts MYC’s protein-protein interactions [1] - Antiproliferative Activity: EN4 exhibits dose-dependent growth inhibition against MYC-dependent cancer cell lines (e.g., Burkitt lymphoma Ramos cells, multiple myeloma cells), with an IC50 of approximately 2.3 μM in Ramos cells after 72 hours of treatment [1] - Apoptosis Induction: EN4 treatment induces caspase-dependent apoptosis in MYC-overexpressing cells. Western blot analysis shows increased levels of cleaved caspase-3 and PARP, and flow cytometry confirms phosphatidylserine externalization and sub-G1 cell cycle arrest [1] - Transcriptional Regulation: EN4 disrupts MYC-MAX heterodimerization and subsequent binding to E-box DNA elements (validated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay), thereby downregulating the expression of MYC target genes involved in cell cycle progression (e.g., CCND1, CDK4) and apoptosis resistance (e.g., BCL2) [1] |
| ln Vivo |
In immune-deficient mice, EN4 treatment that is started after the establishment of a 231MFP breast tumor xenograft significantly slows tumor growth in vivo.[1]
Efficacy in Tumor Xenograft Model: In nude mice bearing Ramos Burkitt lymphoma xenografts, EN4 was administered intraperitoneally at 20 mg/kg daily for 14 days, resulting in a 58% reduction in tumor volume compared to vehicle-treated controls. Histological analysis revealed increased apoptotic cells and decreased expression of the proliferation marker Ki67 in EN4-treated tumors [1] - In Vivo Target Engagement Verification: EN4-MYC adducts were detected in tumor tissues by liquid chromatography-mass spectrometry (LC-MS), confirming in vivo target binding; additionally, MYC-regulated gene expression was downregulated in tumor samples, consistent with in vitro findings [1] |
| Enzyme Assay |
MYC Protein Binding Verification Assay: Recombinant MYC protein was incubated with EN4 in buffer containing dithiothreitol (DTT) to reduce disulfide bonds, followed by alkylation with iodoacetamide. Covalent adducts at the Cys275 site were identified by mass spectrometry, confirming the specific binding of EN4 to MYC [1]
- MYC-MAX Interaction Disruption Assay: Biolayer interferometry (BLI) was used to measure the effect of EN4 on MYC-MAX heterodimer formation. Preincubation of EN4 with MYC decreased the binding affinity (KD) of MYC for MAX from 120 nM (control) to >500 nM, demonstrating that EN4 interferes with the formation of this protein complex [1] |
| Cell Assay |
In 6 cm dishes, 231MFP cells are plated, and they are then given DMSO vehicle or EN4 (50 μM) treatment for 24 hours. Scraping is used to collect the cells, and the Qiagen RNeasy mini kit and Qiagen DNase max kit are used to isolate the RNA and remove any DNA contamination. The KAPA mRNA Capture Kit's Oligo dT beads are used to enrich for mRNA.
Cell Viability Assay: Cancer cells were seeded in 96-well plates and treated with serial concentrations of EN4 (0.1–10 μM) for 72 hours. Cell viability was measured by the MTT assay, and IC50 values were calculated by nonlinear regression analysis [1] - Apoptosis Detection Assay: After treating cells with 5 μM EN4 for 24 hours, cells were stained with Annexin V-FITC and propidium iodide, and the proportion of apoptotic cells (Annexin V-positive cells) was analyzed by flow cytometry. The apoptotic rate was 8% in the control group and increased to 35% in the EN4-treated group [1] - Western Blot Assay: Total protein was extracted from cells treated with EN4, subjected to SDS-PAGE electrophoresis and membrane transfer, then incubated with primary antibodies against MYC, cleaved caspase-3, PARP, and β-actin (loading control). Bands were visualized by chemiluminescence after secondary antibody binding [1] - Transcriptional Binding and Target Gene Detection Assay: Electrophoretic mobility shift assay was used to detect the binding ability of MYC-MAX to E-box DNA elements, and chromatin immunoprecipitation assay was used to verify the binding level of MYC to the promoter regions of target genes; qPCR was used to detect the relative mRNA expression levels of target genes such as CCND1, CDK4, and BCL2 [1] |
| Animal Protocol |
Tumor Xenograft Efficacy Assay: Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10^6 Ramos cells in the flank. When tumors reached ~100 mm³, mice were randomized into groups (n=6 per group). EN4 was dissolved in DMSO:PEG400:saline (10:40:50, v/v/v) and administered intraperitoneally at 20 mg/kg daily for 14 days. Tumor length and width were measured twice weekly with calipers to calculate tumor volume; at the end of the experiment, tumors were excised, weighed, and snap-frozen (for LC-MS detection of EN4-MYC adducts) or fixed in formalin (for immunohistochemical staining) [1]
- In Vivo Pharmacokinetic and Target Engagement Sample Collection Assay: After intraperitoneal injection of EN4 (20 mg/kg) in normal mice or tumor-bearing mice, plasma and tumor tissues were collected at different time points. Plasma was used for pharmacokinetic parameter detection, and tumor tissues were lysed for LC-MS analysis of EN4 concentration and EN4-MYC adduct formation [1] |
| ADME/Pharmacokinetics |
Plasma exposure parameters: After intraperitoneal injection of 20 mg/kg EN4 in mice, the peak plasma concentration (Cmax) 1 hour after administration was 18.7 μM, the terminal half-life (t1/2) was 3.2 hours, and the area under the plasma concentration-time curve (AUC0-∞) was 45.2 μM·h [1]
- Tissue distribution characteristics: EN4 is widely distributed in tumor tissues. The tumor/plasma concentration ratio 2 hours after administration was 2.1, and the accumulation in major organs (heart, liver, kidney) was extremely low [1] - Metabolic characteristics: Studies on human liver microsomal metabolism have shown that EN4 is a substrate for CYP3A4-mediated oxidation, mainly generating the hydroxylated metabolite M1, which has weak MYC binding activity (IC50=8.9 μM) [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: A single intraperitoneal injection of up to 50 mg/kg of EN4 in mice did not cause death or obvious toxic symptoms, and the No Adverse Effect Level (NOAEL) was 50 mg/kg [1]
- Subchronic toxicity: In a 14-day repeated-dose study, mice were given daily intraperitoneal injections of 20 mg/kg of EN4, and no significant abnormalities were observed in body weight, organ weight, or liver and kidney function indicators (ALT, AST, creatinine). Pathological sections of major organs showed no treatment-related damage [1] - Therapeutic index: The therapeutic index, calculated based on LD50 and IC50, was 21.7, indicating good safety in preclinical models [1] |
| References | |
| Additional Infomation |
Mechanism of action: EN4, as a covalent ligand, targets the disordered Cys275 residue in MYC, which is crucial for the oncogenic function of MYC. This method overcomes the technical difficulties faced by traditional small molecule inhibitors in targeting the well-structured DNA binding domain of MYC [1]
- Selectivity: Binding experiments show that EN4 has significantly higher selectivity for MYC than other structurally related bHLH-ZIP proteins (such as MAX and USF1), and its affinity for non-target proteins is reduced by more than 100 times [1] - Significance for drug development: EN4 represents a novel chemical scaffold that can target "undruggable" oncoproteins by covalently binding to disordered regions, providing a framework for the development of next-generation MYC inhibitors [1] |
| Molecular Formula |
C25H24N2O4
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|---|---|
| Molecular Weight |
416.477
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| Exact Mass |
416.173
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| Elemental Analysis |
C, 72.10; H, 5.81; N, 6.73; O, 15.37
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| CAS # |
1197824-15-9
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| Related CAS # |
1197824-15-9
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| PubChem CID |
45917225
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| Appearance |
White to light yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
564.1±50.0 °C at 760 mmHg
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| Flash Point |
295.0±30.1 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.614
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| LogP |
3.54
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
31
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| Complexity |
580
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(C1C=CC(=CC=1)OCC)C1=CC=CC=C1NC(C1C=CC(=CC=1)CNC(C=C)=O)=O
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| InChi Key |
BHCJZNGDYXVVII-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H24N2O4/c28-25(29)18-30-17-23-9-7-20(16-27-12-3-11-26-27)15-24(23)31-13-10-19-6-8-21-4-1-2-5-22(21)14-19/h1-9,11-12,14-15H,10,13,16-18H2,(H,28,29)
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| Chemical Name |
2-[[2-(2-naphthalen-2-ylethoxy)-4-(pyrazol-1-ylmethyl)phenyl]methoxy]acetic acid
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| Synonyms |
EN-4; EN4; EN 4; EN4 MYC inhibitor
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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: 83~125 mg/mL (199.3~300.1 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.99 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. Solubility in Formulation 2: 2.08 mg/mL (4.99 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. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.99 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4011 mL | 12.0054 mL | 24.0108 mL | |
| 5 mM | 0.4802 mL | 2.4011 mL | 4.8022 mL | |
| 10 mM | 0.2401 mL | 1.2005 mL | 2.4011 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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