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| Other Sizes |
| Targets |
RNF114 (IC50 = 470 nM)
RNF114 (Ring Finger protein 114). EN219 is a covalent inhibitor of the E3 ubiquitin ligase RNF114. It binds with moderate specificity to the N-terminal cysteine C8 of RNF114 with an IC50 of 470 nM. It inhibits RNF114-mediated autoubiquitination and p21 ubiquitination. |
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| ln Vitro |
Isotopic tandem orthogonal proteolysis ABPP (isoTOP-ABPP) analysis[1] shows that EN219 (1 μM; 90 min) interacts with RNF114 C8, TUBB1 C201, HSPD1 C442, and HIST1H3A C97.
EN219 is a synthetic covalent ligand with moderate specificity for the N-terminal cysteine C8 of RNF114, with an IC50 of 470 nM. By covalently modifying RNF114, it inhibits the E3 ligase activity of this protein, blocking RNF114-mediated autoubiquitination and the ubiquitination of its substrate p21. This leads to the stabilization of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation. No other specific in vitro potency data are provided. |
| ln Vivo |
In vivo activity data for EN219 are not detailed in the supplier literature. As a covalent inhibitor of RNF114, it has potential applications in studying the role of this E3 ligase in cancer and immune regulation. Further in vivo studies would be required to characterize its efficacy, pharmacokinetics, and safety profile. The covalent binding mechanism suggests that it could have a long-lasting effect.
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| Enzyme Assay |
Non-cellular assays for RNF114 activity involve in vitro ubiquitination reactions using purified recombinant RNF114, an E1 enzyme, an E2 enzyme (e.g., UbcH5c), and ubiquitin. EN219 is pre-incubated with RNF114 (0.1-100 uM) for 30 minutes to allow covalent binding. The reaction is then initiated by adding the other components. The reaction is stopped, and the levels of ubiquitinated RNF114 (autoubiquitination) are detected by Western blot. A reduction in the high-molecular-weight smear indicates inhibition. For binding studies, the mass increase from covalent adduct formation can be monitored by LC-MS.
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| Cell Assay |
Cellular assays involve treating cells (e.g., HEK293T) with EN219 (0.1-50 uM) for 6-24 hours. Cell lysates are analyzed by Western blot for p21 protein levels. An increase in p21 levels indicates successful inhibition of RNF114-mediated p21 ubiquitination and degradation. The compound's effect on global ubiquitination can also be assessed. Cell viability is measured by MTT or CellTiter-Glo to assess any off-target cytotoxicity. Covalent binding can be confirmed by a "click chemistry" approach if an alkyne tag is introduced.
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| Animal Protocol |
For in vivo studies, EN219 could be formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline and administered intraperitoneally to mice. Target engagement can be assessed by performing a Western blot for p21 levels in tissues. Since the compound is a covalent binder, its pharmacodynamic effect (p21 elevation) could be sustained even after the free drug is cleared from the plasma. Detailed PK studies are not described.
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| ADME/Pharmacokinetics |
EN219 is a covalent inhibitor that is cell-permeable. It has a molecular weight of 456.56 g/mol and a molecular formula of C17H13Br2ClN2O. It is soluble in DMSO. The compound is stored at -20degC. Detailed pharmacokinetic parameters (half-life, oral bioavailability) are not reported in the supplier literature. The presence of the reactive chloroacetyl group means it may have off-target reactivity with other proteins and should be handled with care.
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| Toxicity/Toxicokinetics |
Detailed toxicological data for EN219 are not available. It is intended for research use only and not for human consumption. Standard safety precautions for handling research chemicals should be followed, including the use of gloves and eye protection. Because it is a covalent inhibitor, it has the potential for off-target reactivity, and a thorough selectivity profiling would be required to assess its safety in any in vivo context.
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| References | |
| Additional Infomation |
Transforming functionally active natural products into fully synthetic small molecule mimics has always been an important topic in the field of medicinal chemistry. We recently discovered that the terpenoid natural product azadirachtin can be used as a covalent recruiter of the E3 ubiquitin ligase RNF114 for targeting protein degradation—a powerful therapeutic approach in modern drug development. This article reports the discovery of fully synthetic RNF114-based recruiter molecules using an activity-based proteomic analysis covalent ligand screening method. These molecules can also be used for PROTAC applications, and their effectiveness in degrading therapeutically relevant targets (such as BRD4 and BCR-ABL) in cells has been demonstrated. Identifying simple and easy-to-use drug-like scaffolds that can mimic the functions of complex natural products will help to further expand the toolbox of E3 ligase recruiters, which is crucial for drug development and chemical biology. [1]
EN219 is a research-grade chemical, not an FDA-approved drug. It was initially described as a synthetic ligand for RNF114 and represents a valuable tool for chemical biology. It is used to study the ubiquitination of p21 and the regulation of the cell cycle. RNF114 is also a potential target in autoimmune diseases and cancer, making EN219 a useful probe for target validation. The compound is also known as EN219 in the chemical biology literature. |
| Molecular Formula |
C17H13BR2CLN2O
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|---|---|
| Molecular Weight |
456.558921575546
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| Exact Mass |
455.906
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| CAS # |
380351-29-1
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| Related CAS # |
EN219-alkyne
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| PubChem CID |
3413196
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.7
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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 |
3
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| Heavy Atom Count |
23
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| Complexity |
458
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(=O)(N1C(C2=CC=C(Br)C=C2)CC(C2=CC=C(Br)C=C2)=N1)CCl
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| InChi Key |
CXRHMHAKZPJNKS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H13Br2ClN2O/c18-13-5-1-11(2-6-13)15-9-16(22(21-15)17(23)10-20)12-3-7-14(19)8-4-12/h1-8,16H,9-10H2
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| Chemical Name |
1-[3,5-bis(4-bromophenyl)-3,4-dihydropyrazol-2-yl]-2-chloroethanone
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| Synonyms |
EN219; 380351-29-1; 1-[3,5-bis(4-bromophenyl)-3,4-dihydropyrazol-2-yl]-2-chloroethanone; CHEMBL5274261; 3,5-bis(4-bromophenyl)-1-(chloroacetyl)-4,5-dihydro-1H-pyrazole; 1-[3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl]-2-chloroethanone; 1-(3,5-Bis(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-chloroethanone;
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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: 65 mg/mL (142.37 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3.25 mg/mL (7.12 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 DMSO stock solution (32.5 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1903 mL | 10.9515 mL | 21.9029 mL | |
| 5 mM | 0.4381 mL | 2.1903 mL | 4.3806 mL | |
| 10 mM | 0.2190 mL | 1.0951 mL | 2.1903 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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