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Biological Description

Description: E-64 is an irreversible and specific cysteine protease inhibitor. The IC50 of E-64 for papain is 9 nM.

Targets&IC50: Cysteine protease:9 nM

In vitro: E-64 rapidly inhibits the activities of the cysteine proteinases cathepsins B, H and L and papain, while has no effect on the serine proteinases or the metalloproteinases. E-64, as a specific inhibitor os cysteine proteases, effectively blocks in vitro ovarian cancer cell invasion. In addition, E-64 also shows antiparasitic activity against Giardia lamblia excystation in vitro. E-64 improves the preimplantation development of bovine somatic cell nuclear transfer embryos, which indicates another important implication of E-64. 

In vivo: E-64 induces a marked decrease in the number of spontaneous metastasis in M5076 tumor bearing mice, while has no effect on the formation of experimental metastasis. E-64 also shows antiparasitic activity against Giardia lamblia excystation in infected mice.

Kinase Assay: The Cathepsin Β 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.

Chemical Properties

Molecular Weight: 357.411

Formula: C15H27N5O5

CAS No.: 66701-25-5

Storage & Solubility Information

Storage

Powder: -20°C for 3 years

In solvent: -80°C for 2 years

Solubility Information

H2O: 9 mg/mL (25.2 mM)

Ethanol: <1 mg/mL

DMSO: 66 mg/mL (184.7 mM)

( < 1 mg/ml refers to the product slightly soluble or insoluble )

Catalog #: T6037

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References and Literature

1. Matsumoto K, et al. Biopolymers. 1999, 51(1), 99-107.

2. Barrett AJ, et al. Biochem J. 1982, 201(1), 189-198.

3. Kobayashi H, et al. Cancer Res. 1992, 52(13), 3610-3614.

4. Hussein EM, et al. J Egypt Soc Parasitol. 2009, 39(1), 111-119.

5. Min SH, et al. J Reprod Dev. 2014, 60(1), 21-27.

6. Blass G, et al. Chronic cathepsin inhibition by E-64 in Dahl salt-sensitive rats. Physiol Rep. 2016 Sep;4(17). pii: e12950.

7. Zhang B, Chen Z. Screening of cathepsin B inhibitors in traditional Chinese medicine by capillary electrophoresis with immobilized enzyme microreactor[J]. Journal of Pharmaceutical and Biomedical Analysis. 2019: 112811.

8. Jiang T Y, Pan Y F, Wan Z H, et al. PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma[J]. Science Translational Medicine. 2020, 12(562).