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E-64

E-64

Catalog No.

A2576

cysteine proteases inhibitor

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Grouped product items
Size	Price	Stock
10mM (in 1mL DMSO)	$55.00	In stock
Evaluation Sample	$30.00	In stock
5mg	$50.00	In stock
25mg	$140.00	In stock
100mg	$420.00	In stock
250mg	$980.00	In stock

For scientific research use only and should not be used for diagnostic or medical purposes.

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Background
Product Citation
Chemical Properties
Protocol
Biological Activity
Quality Control

Background

E-64 (CAS 66701-25-5) is a selective inhibitor targeting cysteine proteases, isolated originally from Aspergillus cultures. Structurally classified as an L-trans-epoxysuccinyl peptide, it irreversibly inhibits proteases such as papain, ficin, and bromelain by covalent binding to the active-site cysteine residue. Its inhibitory activity additionally extends to mammalian cysteine proteases including cathepsins B, H, and L as well as calcium-dependent protease calpain, with reported IC50 values ranging approximately between 10-100 nM depending on specific enzyme assays and conditions. In research laboratories, E-64 is primarily utilized for mechanistic studies, active-site titration assays, and quantitative evaluation of cysteine protease concentrations and enzyme kinetics.

References:
1. A. J. BARRETT, A. A. KEMBHAVI, L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem. J. (1982) 201, 189-198
2. Hanada, K., Tamai, M., Yamagishi, M., Ohmura, S., Sawada, J. & Tanaka, I. (1978c) Agric. Biol. Chem. 42, 523-528
3. Towatari, T., Tanaka, K., Yoshikawa, D. & Katunuma, N. (1978).J. Biochem. (Tokyo) 84, 659-671.
4. Mort, J. S., Recklies, A. D. & Poole, A. R. (1980) Biochim. Biophys. Acta 614, 134-143.
5. Sugita, H., Ishiura, S., Suzuki, K. & Imahori, K. (1980) J. Biochem. (Tokyo) 87, 339-341
6. Hanada, K., Tamai, M., Morimoto, S., Adachi, T.,Ohmura, S., Sawada, J. & Tanaka, I. (1978a) Agric. Biol. Chem. 42, 537-541.
7. Hanada, K., Tamai, M., Ohmura, S., Sawada, J., Seki, T.& Tanaka, I. (1978b)Agric. Biol. Chem. 42, 529-536
8. Knight, C. G. (1980) Biochem. J. 189,447-453
9. Takio, K., Towatari, T., Katunuma, N. & Titani, K.(1980) Biochem. Biophys. Res. Commun. 97, 340-346
10. Bender, M. L., Begue-Canton, M. L., Blakeley, R. L.,Brubacher, L. J., Feder, J., Gunter, C. R., Kezdy, F. J.,Killheffer, J. V., Marshall, T. H., Miller, C. G., Roeske,R. W. & Stoops, J. K. (1966) J. Am. Chem. Soc. 88,5890-5913

Product Citation

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Chemical Properties

Physical Appearance	A solid
Storage	Store at -20°C
M.Wt	357.41
Cas No.	66701-25-5
Formula	C₁₅H₂₇N₅O₅
Solubility	≥49.1 mg/mL in H2O; ≥53.6 mg/mL in DMSO; ≥55.2 mg/mL in EtOH
Chemical Name	(2S,3S)-3-[[(2S)-1-[4-(diaminomethylideneamino)butylamino]-4-methyl-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylic acid
SDF	Download SDF
Canonical SMILES	CC(C)CC(C(=O)NCCCCN=C(N)N)NC(=O)C1C(O1)C(=O)O
Shipping Condition	Small Molecules with Blue Ice, Modified Nucleotides with Dry Ice.
General tips	We do not recommend long-term storage for the solution, please use it up soon.

Protocol

Cell experiment: [1]
Cell lines	H-59 and M-27 cells
Preparation method	The solubility of this compound in DMSO is ≥53.6 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37°C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20°C for several months.
Reaction Conditions	10 μg/mL, 48 hours
Applications	E-64 inhibited H-59 invasion in a dose-dependent manner with a maximal inhibition of 97% at a concentration of 10 μg/mLwhich was non-toxic. Cell migration as measured with filters coated with 7.5 μg/filter type IV collagen was reduced by only 25% suggesting that the cysteine proteinases played a more minor role in cell migration in the absence of a basement membrane barrier. On the other hand M-27 invasion was not significantly affected by treatment with E-64 even at concentrations as high as 100 μg/mL.
Animal experiment: [2]
Animal models	Wistar strain rats
Dosage form	Intraperitoneal injection, 1 mg/100 g body weight
Applications	The animals were killed 1 h after the injection and the cathepsin B and cathepsin L activities in the lysosomal were assayed. The inhibition caused by E-64 was already detectable 1 hour after its injection.
Other notes	Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.
References: [1] Navab R, Mort J S, Brodt P. Inhibition of carcinoma cell invasion and liver metastases formation by the cysteine proteinase inhibitor E-64. Clinical & experimental metastasis, 1997, 15(2): 121-129. [2] Hashida S, TOWATARI T, KOMINAMI E, et al. Inhibitions by E-64 derivatives of rat liver cathepsin B and cathepsin L in vitro and in vivo. Journal of biochemistry, 1980, 88(6): 1805-1811.

Biological Activity

E-64 is a natural, potent, and irreversible inhibitor of cysteine proteases.Its IC50 values for inhibiting cathepsins K, S, and L, in vitro, are 1.4, 4.1, and 2.5 nM, respectively.
Targets	cathepsins K	cathepsins S	cathepsins L
IC50	1.4 nM	4.1 nM	2.5 nM