E-64
Based on 23 publication(s) in Google Scholar
E-64 (Proteinase inhibitor E 64) is a potent irreversible inhibitor against general cysteine proteases with IC50 of 9 nM for papain.
For research use only. We do not sell to patients.
- Purity: 99.95%
- CAS No.: 66701-25-5
- Formula: C15H27N5O5
- Molecular Weight:357.41
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Storage:Powder -20°C, 3 years ; In solvent -80°C, 6 months , -20°C, 1 month
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
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Publications Citing Use of MedChemExpress (MCE) E-64
More- Signal Transduct Target Ther. 2025 Dec 15;10(1):406. [Abstract]
- Nat Biotechnol. 2025 Sep 10. [Abstract]
- J Extracell Vesicles. 2025 Jan;14(1):e70041. [Abstract]
- Adv Sci (Weinh). 2025 Aug 7:e09208. [Abstract]
- Sci Adv. 2025 Sep 26;11(39):eadx3827. [Abstract]
- Phytomedicine. 2025 Apr 3:141:156720. [Abstract]
- Cell Prolif. 2019 May;52(3):e12609. [Abstract]
- Int J Oncol. 2019 Jul;55(1):331-339. [Abstract]
- LWT-FOOD SCI TECHNOL. 2018 Jan, 87; 186-193
- Protein Sci. 2024 Dec;33(12):e5225. [Abstract]
- Int J Mol Sci. 2023 Apr 17;24(8):7406. [Abstract]
- Am J Respir Cell Mol Biol. 2025 Feb;72(2):145-157. [Abstract]
- PLoS Pathog. 2025 Oct 17;21(10):e1013581. [Abstract]
- Sci Rep. 2022 Jul 16;12(1):12197. [Abstract]
- PLoS Pathog. 2022 Jan 14;18(1):e1010204. [Abstract]
- Virol Sin. 2023 Dec;38(6):911-921. [Abstract]
- Curr Issues Mol Biol. 2025 May 8;47(5):338. [Abstract]
- J Immunol. 2026 Mar 17;215(3):vkag020. [Abstract]
- FEBS Lett. 2021 Jan;595(2):169-182. [Abstract]
- Dev Comp Immunol. 2018 Jan:78:114-123. [Abstract]
- J Aquat Food Prod T. 2019, 28(6), 658–666.
- Acta Crystallogr F Struct Biol Commun. 2025 Feb 1;81(Pt 2):53-61. [Abstract]
- bioRxiv. 2026 Mar 5:2026.03.04.709643. [Abstract]
Customer Validation & Images
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Cell Proliferation/Viability Assay
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Others
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Flow Cytometry
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Flow Cytometry
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Cell Proliferation/Viability Assay
All Cathepsin Isoforms
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Biological Activity
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cathepsin K |
cathepsin L |
E-64 (Proteinase inhibitor E 64) is a cathepsin B-specific inhibitor, and its binding modes with papain, actinidin, cathepsin L, and cathepsin K have been reviewed at the atomic level. E-64 has been widely used as a potent and irreversible (covalent-type) inhibitor for many cysteine proteases such as papain, ficin, actinidin, cathepsin B and L[1]. The S.cervi adult parasites are incubated in the Kreb's Ringer bicarbonate (KRB) maintenance medium for 8 h at 37°C, 5% CO2 with 5, 10, 20 and 40 μM concentration of E-64. E-64 shows a concentration and time dependent decrease in motility and viability of the parasites (EC50=16 μM)[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Chemical Information
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CAS No. 66701-25-5
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Appearance Solid
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Molecular Weight 357.41
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Formula C15H27N5O5
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Color White to off-white
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SMILES
O=C([C@H]1O[C@@H]1C(N[C@H](C(NCCCCNC(N)=N)=O)CC(C)C)=O)O
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Synonyms
Proteinase inhibitor E 64
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Shipping
Room temperature in continental US; may vary elsewhere.
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Storage
Powder -20°C 3 years In solvent -80°C 6 months -20°C 1 month
Publications (23)
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Journal Impact Factor
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Most Recent
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Signal Transduct Target Ther
Selective depletion of tumor-associated SAMHD1 enhances chemotherapeutic efficacy and antitumor immune responses. [Abstract]2025 Dec 15;10(1):406. PMID: 41392286 -
Nat Biotechnol
A rapid imaging-based screen for induced-proximity degraders identifies a potent degrader of oncoprotein SKP2. [Abstract]2025 Sep 10. PMID: 40931108 -
J Extracell Vesicles
Extracellular Vesicles From Bone Marrow-Derived Macrophages Enriched in ARG1 Enhance Microglial Phagocytosis and Haematoma Clearance Following Intracerebral Haemorrhage. [Abstract]2025 Jan;14(1):e70041. PMID: 39868438 -
Adv Sci (Weinh)
2025 Aug 7:e09208. PMID: 40776529
E-64 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2025 Aug 7:e09208. [Abstract]
CT26 cells were treated with E-64 (10 μg/mL; 12 h) and cell viability was then assessed.
E-64 purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2025 Aug 7:e09208. [Abstract]
After treating CT26 cells with E-64 (10 μg/mL; 12 h), the release of lactate dehydrogenase (LDH) was detected.
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Sci Adv
USP13 stabilizes NLRP3 to facilitate inflammasome activation by preventing TRIM31-mediated NLRP3 ubiquitination and degradation. [Abstract]2025 Sep 26;11(39):eadx3827. PMID: 41004574 -
Phytomedicine
Sophoraflavanone G as an ectosteric inhibitor of cathepsin K attenuates ovariectomy-induced bone loss by suppressing bone resorption. [Abstract]2025 Apr 3:141:156720. PMID: 40220429 -
Cell Prolif
Inhibition of PI3K/mTOR increased the sensitivity of hepatocellular carcinoma cells to cisplatin via interference with mitochondrial-lysosomal crosstalk. [Abstract]2019 May;52(3):e12609. PMID: 31033054
E-64 purchased from MedChemExpress. Usage Cited in: Cell Prolif. 2019 May;52(3):e12609. [Abstract]
Huh7 cells were treated with 8 μg/mL cisplatin and/or 5 μmol/L PKI‐402 in the presence or absence of 5 μmol/L E‐64 for 24 h and then stained with Annexin V and PI and analysed by FlowJo.
E-64 purchased from MedChemExpress. Usage Cited in: Cell Prolif. 2019 May;52(3):e12609. [Abstract]
Huh7 cells were treated with 8 μg/mL cisplatin and/or 5 μmol/L PKI‐402 in the presence or absence of 5 μmol/L E‐64 for 24 h and then stained with MitoSOX Red and detected using flow cytometry.
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Int J Oncol
Metformin induces TPC-1 cell apoptosis through endoplasmic reticulum stress-associated pathways in vitro and in vivo. [Abstract]2019 Jul;55(1):331-339. PMID: 31180536 -
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Protein Sci
Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome. [Abstract]2024 Dec;33(12):e5225. PMID: 39589076 -
Int J Mol Sci
Modifying the Expression of Cysteine Protease Gene PCP Affects Pollen Development, Germination and Plant Drought Tolerance in Maize. [Abstract]2023 Apr 17;24(8):7406. PMID: 37108569 -
Am J Respir Cell Mol Biol
Airway Epithelium-derived CXCL14 Promotes Eosinophil Accumulation in Allergic Airway Inflammation. [Abstract]2025 Feb;72(2):145-157. PMID: 39141567 -
PLoS Pathog
HBV p22-interacting protein C1QBP inhibits viral replication through impeding nucleocapsid formation and nuclear import. [Abstract]2025 Oct 17;21(10):e1013581. PMID: 41105696 -
Sci Rep
Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease. [Abstract]2022 Jul 16;12(1):12197. PMID: 35842458 -
PLoS Pathog
Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy. [Abstract]2022 Jan 14;18(1):e1010204. PMID: 35030230 -
Virol Sin
African swine fever virus S273R protein antagonizes type I interferon production by interfering with TBK1 and IRF3 interaction. [Abstract]2023 Dec;38(6):911-921. PMID: 37659477 -
Curr Issues Mol Biol
Mechanistic Insights into the Anti-Hepatocellular Carcinoma Effects of ACY-1215: p53 Acetylation and Ubiquitination Regulation. [Abstract]2025 May 8;47(5):338. PMID: 40699737 -
J Immunol
Cathepsin B promotes asthma potentially via macrophage-associated autophagy and apoptosis. [Abstract]2026 Mar 17;215(3):vkag020. PMID: 41847863 -
FEBS Lett
ABRO1 stabilizes the deubiquitinase BRCC3 through inhibiting its degradation mediated by the E3 ubiquitin ligase WWP2. [Abstract]2021 Jan;595(2):169-182. PMID: 33107021 -
Dev Comp Immunol
Cathepsin L-like protease can regulate the process of metamorphosis and fat body dissociation in Antheraea pernyi. [Abstract]2018 Jan:78:114-123. PMID: 28958702
E-64 purchased from MedChemExpress. Usage Cited in: Dev Comp Immunol. 2018 Jan:78:114-123. [Abstract]
The specific activity of the Ap-cathL. E64 (10 mM; 8 h) is used to inhibit the Ap-cathL activitys.
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Acta Crystallogr F Struct Biol Commun
Structural insights into the role of the prosegment binding loop in a papain-superfamily cysteine protease from Treponema denticola. [Abstract]2025 Feb 1;81(Pt 2):53-61. PMID: 39846228 -
bioRxiv
Comparative Genomic and Functional Profiling of ECM-Targeting Enzymes in Bacteroides, a Key Genus of the Human Gut Microbiome. [Abstract]2026 Mar 5:2026.03.04.709643. PMID: 41847005
Solvent & Solubility
DMSO : 58.33 mg/mL (163.20 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
H2O : 5 mg/mL (13.99 mM; Need ultrasonic)
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
Select the appropriate dissolution method based on your experimental animal and administration route.
- For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
- To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for In Vivo experiments, it is recommended to prepare freshly and use it on the same day.
- The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.
Add each solvent one by one: 10% DMSO 90% Saline
Solubility: ≥ 2.5 mg/mL (6.99 mM); Clear solution
Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
Solubility: ≥ 2.08 mg/mL (5.82 mM); Clear solution
This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.
Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: ≥ 2.08 mg/mL (5.82 mM); Clear solution
This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
For the following dissolution methods, please prepare the working solution directly:
It is recommended to prepare fresh solutions and use them promptly within a short period of time.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.
Add each solvent one by one: PBS
Solubility: 50 mg/mL (139.90 mM); Clear solution; Need ultrasonic
Please enter the basic information of animal experiments:
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Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
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%DMSO +
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
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%+
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+%Tween-80 + +
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%Saline +
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Working solution concentration: 0.22 mg/mL
Method for preparing stock solution: mg drug dissolved in μL DMSO. Stock solution concentration: mg/mL.
1. Take μL DMSO stock solution;
2. Add μL .
μL , mix evenly;
3. Then add μL Tween 80, mix evenly;
4. Then add μL
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Protocol
The Cathepsin B activity is determined using Z-Arg-Arg-4mβNA as substrate with slight modifications. The crude extract is pre-incubated at 37°C for 5 min in 50 mM sodium acetate buffer, pH 5.0 containing 1 mM EDTA and 5 mM DTT. The substrate (final concentration, 100 μM) is added to make the final assay volume of 1 mL. The reaction mixture is incubated at 37°C for 30 min. The reaction is terminated by adding equal volume of stopping reagent containing Fast Garnet GBC salt (1 mg/mL), 10 mM pHMB and 50 mM EDTA, pH 6.0. The extraction of product, β-napthylamine (β-NA), is carried out with n-butanol. After complete layer separation, the absorbance is measured in n-butanol layer and activity is calculated using molar extinction coefficient of β-napthylamine solution as 31.5 M/cm per sec at 520 nm. One unit of enzyme activity is defined as the amount of enzyme liberating 1 μmol of βNA per minute at 37°C. The half maximal inhibitory concentration (IC50) is calculated by plotting the graph between the different concentration of E-64 and the % inhibition in cathepsin B activity. Here, IC50 indicates the concentration of the E-64 required to inhibit the parasitic cathepsin B activity by half[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Mice[3]
The NOD/LtJ and BDC2.5 T cell receptor (TCR) transgenic NOD mice are used to study the effects of treatment with anti-serpin B13 monoclonal antibody (mAb). Four-week-old female NOD/LtJ mice are injected intravenously four times over a period of 10 days with anti-serpin B13 mAb (100 μg/injection). In addition, during the same period, some animals are also injected intraperitoneally with the protease inhibitor E64 at 10 mg/kg/day for several days. Control mice are treated with diluent (a sterilized PBS solution containing 10% DMSO) and control IgG. The solutions containing E64 or DMSO are prepared immediately before use. Twenty-four hours after the last injection, the mice are killed, and cells from their lymphoid organs and pancreatic islets are subjected to FACS analysis.
Rats[4]
Seven-week old male Dahl Salt Sensitive rats (SS/JrHsdMcwi) are used. Briefly, 8-week old anesthetized SS rats have their left femoral artery and vein catheterized. Both catheters are fixed and exteriorized from the back of the neck and the arterial line is connected to a heparinized saline infusion pump that is in line with a blood pressure transducer, and the venous line is connected to a saline infusion pump. Animals are allowed 360° movement using a tether-swivel system. This preparation allowed chronic venous infusion and arterial blood pressure measurement in conscious, freely moving rats. A stable baseline blood pressure is obtained for 4 days prior to switching both groups to an 8.0% NaCl diet and the simultaneous addition of E-64 (1 mg/day; 280 mM stock in DMSO) or the vehicle (DMSO in saline) control to the venous catheter. Daily MAP is calculated by averaging MAP taken every min over the beginning 3 h period of the rat sleep cycle.
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Purity & Documentation
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Data Sheet (278 KB)
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SDS (419 KB)
- English - EN (419 KB)
- Français - FR (419 KB)
- Deutsch - DE (419 KB)
- Norwegian - NO (419 KB)
- Español - ES (419 KB)
- Swedish - SV (419 KB)
- Italian - IT (419 KB)
- Korean - KR (419 KB)
- Portuguese - PT (419 KB)
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Handling Instructions (2659 KB)
References
[1]. Matsumoto K, et al. Structural basis of inhibition of cysteine proteases by E-64 and its derivatives. Biopolymers. 1999;51(1):99-107. [Content Brief]
[2]. Wadhawan M, et al. Inhibition of cathepsin B by E-64 induces oxidative stress and apoptosis in filarial parasite. PLoS One. 2014 Mar 25;9(3):e93161. [Content Brief]
[3]. Baldzizhar R, et al. Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes. J Biol Chem. 2013 Jan 18;288(3):1612-9. [Content Brief]
[4]. Blass G, et al. Chronic cathepsin inhibition by E-64 in Dahl salt-sensitive rats. Physiol Rep. 2016 Sep;4(17). pii: e12950. [Content Brief]
Complete Stock Solution Preparation Table
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
| Optional Solvent | Concentration Solvent Mass | 1 mg | 5 mg | 10 mg | 25 mg |
|---|---|---|---|---|---|
| H2O / DMSO | 1 mM | 2.7979 mL | 13.9895 mL | 27.9791 mL | 69.9477 mL |
| 5 mM | 0.5596 mL | 2.7979 mL | 5.5958 mL | 13.9895 mL | |
| 10 mM | 0.2798 mL | 1.3990 mL | 2.7979 mL | 6.9948 mL | |
| DMSO | 15 mM | 0.1865 mL | 0.9326 mL | 1.8653 mL | 4.6632 mL |
| 20 mM | 0.1399 mL | 0.6995 mL | 1.3990 mL | 3.4974 mL | |
| 25 mM | 0.1119 mL | 0.5596 mL | 1.1192 mL | 2.7979 mL | |
| 30 mM | 0.0933 mL | 0.4663 mL | 0.9326 mL | 2.3316 mL | |
| 40 mM | 0.0699 mL | 0.3497 mL | 0.6995 mL | 1.7487 mL | |
| 50 mM | 0.0560 mL | 0.2798 mL | 0.5596 mL | 1.3990 mL | |
| 60 mM | 0.0466 mL | 0.2332 mL | 0.4663 mL | 1.1658 mL | |
| 80 mM | 0.0350 mL | 0.1749 mL | 0.3497 mL | 0.8743 mL | |
| 100 mM | 0.0280 mL | 0.1399 mL | 0.2798 mL | 0.6995 mL |
* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.
E-64 Related Classifications
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