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N-Ethylmaleimide (also known as NEM) is a naturally occurring biochemical substance that is produced from maleic acid. The mechanism of action of this irreversible inhibitor of cysteine peptidases is to alkylate the enzymes at the thiol group. Enzymes called cysteine proteases, or thiol proteases, break down proteins. A catalytic triad or dyad comprising a nucleophilic cysteine thiol is the common catalytic mechanism between them. Cysteine proteases are found in many fruits, such as kiwifruit, papaya, pineapple, and figs. When fruit is not fully ripe, the amount of protease in it usually increases. Indeed, cysteine proteases have been found in dozens of latices belonging to various plant families. Meat tenderizers include cysteine proteases as an ingredient.
Physicochemical Properties
| Molecular Formula | C6H7NO2 | |
| Molecular Weight | 125.13 | |
| Exact Mass | 125.047 | |
| Elemental Analysis | C, 57.59; H, 5.64; N, 11.19; O, 25.57 | |
| CAS # | 128-53-0 | |
| Related CAS # | N-Ethylmaleimide-d5;360768-37-2 | |
| PubChem CID | 4362 | |
| Appearance | White to light yellow solid powder | |
| Density | 1.2±0.1 g/cm3 | |
| Boiling Point | 210.0±9.0 °C at 760 mmHg | |
| Melting Point | 43-46 °C(lit.) | |
| Flash Point | 73.3±0.0 °C | |
| Vapour Pressure | 0.2±0.4 mmHg at 25°C | |
| Index of Refraction | 1.514 | |
| LogP | 0.68 | |
| Hydrogen Bond Donor Count | 0 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 1 | |
| Heavy Atom Count | 9 | |
| Complexity | 166 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C1C([H])=C([H])C(N1C([H])([H])C([H])([H])[H])=O |
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| InChi Key | HDFGOPSGAURCEO-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3 | |
| Chemical Name | 1-ethylpyrrole-2,5-dione | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| 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. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Prolyl endopeptidase (IC50 = 6.3 μM)； Sulfhydryl (-SH) groups of proteins [2] |
| ln Vitro |
N-Ethylmaleimide (20 μM;30 min) prevents the phosphorylation of Akt Ser-473, Akt Thr-308, p70S6K, ribosomal protein S6, 4E-BP1, eIF4E, BAD, and FKHR-L1[2]. N-Ethylmaleimide (20 μM;30 min) affects conversion of pro-caspase-3 in vascular smooth muscle cells[2]. N-Ethylmaleimide (20 μM;6 h) induces apoptosis in vascular smooth muscle cells[2]. N-Ethylmaleimide (20 μM;30 min) has an impact on ROS production and PP2A activity in vascular smooth muscle cells[2]. N-ethylmaleimide (NEM) was used to block sulfhydryl groups in cell experiments. Treatment with NEM (100 μM) inhibited the neddylation process, as indicated by the accumulation of unneddylated Cullin1 in HEK293T cells. This inhibition affected the β-catenin degradation pathway in the Wnt signaling, leading to the accumulation of β-catenin and increased expression of its target genes such as AXIN2 and MYC [1] |
| ln Vivo |
NEM (10 mg/kg) was administered intraperitoneally to rats, and it reversed the gastroprotective effect of momordin Ic on ethanol-induced gastric mucosal lesions. This reversal was associated with the depletion of sulfhydryl groups, as NEM reacts with sulfhydryl groups to form covalent adducts, thereby inhibiting the protective role of endogenous sulfhydryls in gastric mucosa [2] N-Ethylmaleimide (10 mg/kg; i.h.) increases the prevalence situation in mice with acute gastric ulcers[3]. |
| Cell Assay |
HEK293T cells were seeded and cultured, then treated with NEM (100 μM) for a certain period. Cell lysates were prepared, and Western blot analysis was performed to detect the levels of neddylated and unneddylated Cullin1, as well as β-catenin and its target gene products (AXIN2, MYC). The results showed that NEM inhibited neddylation and affected β-catenin degradation [1] Cell lysates are prepared in Pierce IP Lysis Buffer, which contains protease inhibitors, phosphatase inhibitors, and the deubiquitinating inhibitor N-Ethylmaleimide (NEM, 10mM), in order to facilitate endogenous protein immunoprecipitation. The lysate supernatant is first incubated for 14 hours at 4 °C with a primary antibody, and then it is combined with Dynabeads Protein G magnetic beads and left to rotate for 4 hours at 4 °C. After using a magnetic separation rack to separate the magnetic bead-Ab complex, PBST buffer is used three times to wash the beads. The complex-bound beads are resuspended in 1x protein electrophoresis loading buffer and heated to 100 °C for 8 minutes, after which the supernatant is collected for SDS-PAGE analysis. Before preparing cell lysates for exogenous protein immunoprecipitation, cells are transfected for 48 hours. Antibody against mouse IgG is used in parallel to prepare the negative control. |
| Animal Protocol |
Rats were fasted for 24 hours before the experiment. NEM was dissolved in a suitable solvent and administered intraperitoneally at a dose of 10 mg/kg. After 30 minutes, momordin Ic was given orally, followed by ethanol administration 30 minutes later to induce gastric mucosal lesions. The rats were sacrificed after a certain period, and the gastric mucosa was examined for lesion formation [2] Male Sprague-Dawley rats 10 mg/kg s.c. |
| Toxicity/Toxicokinetics |
Adverse Effects
Dermatotoxin - Skin burns. Lacrimator (Lachrymator) - A substance that irritates the eyes and induces the flow of tears. Toxic Pneumonitis - Inflammation of the lungs induced by inhalation of metal fumes or toxic gases and vapors. 4362 rat LD50 oral 25 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD National Technical Information Service., OTS0536969 4362 rat LD50 intraperitoneal 1 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY) National Technical Information Service., OTS0536969 4362 rat LDLo intracrebral 1669 ug/kg Journal of Medicinal Chemistry., 15(534), 1972 4362 mouse LD50 oral 25 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY) National Technical Information Service., OTS0536969 4362 mouse LD50 intraperitoneal 5 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY) National Technical Information Service., OTS0536969 |
| References |
[1]. Neddylation is essential for β-catenin degradation in Wnt signaling pathway. Cell Rep. 2022 Mar 22;38(12):110538. [2]. Roles of capsaicin-sensitive sensory nerves, endogenous nitric oxide, sulfhydryls, and prostaglandins in gastroprotection by momordin Ic, an oleanolic acid oligoglycoside, on ethanol-induced gastric mucosal lesions in rats. Life Sci. 1999;65(2):PL27-32. |
| Additional Infomation |
N-ethylmaleimide is a member of the class of maleimides that is the N-ethyl derivative of maleimide. It has a role as an EC 2.1.1.122 [(S)-tetrahydroprotoberberine N-methyltransferase] inhibitor, an EC 2.7.1.1 (hexokinase) inhibitor, an EC 1.3.1.8 [acyl-CoA dehydrogenase (NADP(+))] inhibitor and an anticoronaviral agent. It is functionally related to a maleimide. A sulfhydryl reagent that is widely used in experimental biochemical studies. β-Catenin is a central component in the Wnt signaling pathway; its degradation has been tightly connected to ubiquitylation, but it is rarely examined by loss-of-function assays. Here we observe that endogenous β-catenin is not stabilized upon ubiquitylation depletion by a ubiquitylation inhibitor, TAK-243. We demonstrate that N-terminal phosphorylated β-catenin is quickly and strongly stabilized by a specific neddylation inhibitor, MLN4924, in all examined cell types, and that β-catenin and TCF4 interaction is strongly enhanced by inhibition of neddylation but not ubiquitylation. We also confirm that the E3 ligase β-TrCP2, but not β-TrCP1, is associated with neddylation and destruction of β-catenin. GSK3β and adenomatous polyposis coli (APC) are not required for β-catenin neddylation but essential for its subsequent degradation. Our findings not only clarify the process of β-catenin modification and degradation in the Wnt signaling pathway but also highlight the importance of reassessing previously identified ubiquitylation substrates. [1] The roles of capsaicin-sensitive sensory nerves (CPSN), endogenous nitric oxide (NO), sulfhydryls (SHs), prostaglandins (PGs) in the gastroprotection by momordin Ic, an oleanolic acid oligoglycoside isolated from the fruit of Kochia scoparia (L.) SCHRAD., on ethanol-induced gastric mucosal lesions were investigated in rats. Momordin Ic (10 mg/kg, p.o.) potentially inhibited ethanol-induced gastric mucosal lesions. The effect of momordin Ic was markedly attenuated by the pretreatment with capsaicin (125 mg/kg in total, s.c., an ablater of CPSN), N(G)-nitro-L-arginine methyl ester (L-NAME, 70 mg/kg, i.p., an inhibitor of NO synthase), N-ethylmaleimide (NEM, 10 mg/kg, s.c., a blocker of SHs), or indomethacin (10 mg/kg, s.c., an inhibitor of PGs biosynthesis). The attenuation of L-NAME was abolished by L-arginine (300 mg/kg, i.v., a substrate of NO synthase), but not by D-arginine (300 mg/kg, i.v., the enatiomer of L-arginine). The effect of the combination of capsaicin with indomethacin, NEM, or L-NAME was not more potent than that of capsaicin alone. The combination of indomethacin and NEM, indomethacin and L-NAME, or indomethacin and NEM and L-NAME increased the attenuation of each alone. These results suggest that CPSN play an important role in the gastroprotection by momordin Ic on ethanol-induced gastric mucosal lesions, and endogenous PGs, NO, and SHs interactively participate, in rats. [2] - NEM is a sulfhydryl-reactive compound that covalently modifies free sulfhydryl groups in proteins, thereby inhibiting their function. In the context of Wnt signaling, it affects neddylation by targeting sulfhydryl groups in relevant proteins, disrupting β-catenin degradation [1] - In studies on gastric mucosal protection, NEM is used as a tool to deplete endogenous sulfhydryl groups, helping to investigate the role of sulfhydryls in gastroprotective mechanisms [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (16.62 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 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 (16.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (16.62 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 100 mg/mL (799.17 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 7.9917 mL | 39.9584 mL | 79.9169 mL | |
| 5 mM | 1.5983 mL | 7.9917 mL | 15.9834 mL | |
| 10 mM | 0.7992 mL | 3.9958 mL | 7.9917 mL |