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Definition
L-trans-epoxysuccinyl peptide is a potent inhibitor specific for cathepsin B in vitro (IC50= 2.24 nM for rat liver cathepsin B) and in vivo. L-trans-epoxysuccinyl-leucylamido (4-guanidino)butane (E-64) rapidly in activates the cysteine proteinases cathepsins B, H and L and papain.

Discovery
In 1982, Barrette et al., identified a new class of compounds that show promise of acting as class-specific inhibitors for the cysteine proteinases, are the L-trans-epoxysuccinylpeptides related to the compound E-64 [L-trans-epoxysuccinyl- L-leucylamido(4-guanidino)butanel, isolated from cultures of Aspergillus japonicus 1, 2.

Structural Characteristics
[L-3-trans-Carboxyoxiran-2-Carbonyl]-L-Leu-Agmatin][N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide] has a structure C15H27N5O5, 0.5 H2O and molecular weight of 357.41 kDa 3.

Mode of Action
E-64 was shown to inhibit papain, ficin and the fruit and stem bromelains, with disappearance of the thiol group of papain. In contrast, the serine proteinases trypsin, chymotrypsin, tissue kallikrein, plasmin and pancreatic elastase, and the aspartic proteinases pepsin and Paecilomyces acid proteinase are unaffected. E-64 does not inhibit all of the cysteine proteinases. E-64 inactivates cysteine proteinases papain and rat cathepsins B and L by stoichiometric reaction with the cysteine residue essential for catalytic activity. Lineweaver-Burk plots of inhibition data seemed to show that the action of E-64 was not competitive with substrate 1, 4. Effective irreversible inhibitor of cysteine proteases. E-64 acts by forming a thioether bond with the thiol of the active cysteine. This compound is of the epoxysuccinate class of inhibitors, which affect primarily cysteine peptidases. The compounds inhibit irreversibly by S-alkylation of the catalytic cysteine, which results in opening of the epoxide ring. A thioester bond is formed by nucleophilic attack at C2 or C3 of the epoxide ring 3.

Functions

Inhibition of two mammalian cysteine proteinases:
E-64 has been reported to inhibit two other mammalian cysteine proteinases: cathepsin L and a proteinase from human breast-tumour tissue and the calcium-dependent proteinase, calpain, from chicken muscle 5, 6.

As a potent hypocalcemic agents: Epoxysuccinyl amino acid derivatives an object of the present invention to provide a cysteine proteinase inhibitor which is a potent hypocalcemic agent when given orally or intravenously. It is also an object of the present invention to provide an orally active agent for the treatment of diseases associated with increased bone resorption. It is also an object of the present invention to treat diseases associated with increased osteolysis, such as early stages of malignant hypercalcemia, Paget's disease and osteoporosis.

Effective against bacterial infection: Abnormally down-regulated protein kinase C (PKC) activity is responsible for the impaired cellular function of natural killer cells and polymorphonuclear cells, and the giant granule formation in fibroblasts in the beige mouse, an animal model of Chediak-Higashi syndrome. Oral or intraperitoneal administration of E-64, protects PKC from calpain-mediated proteolysis, on the impaired cellular function in PMNs from beige mice. In addition, E-64 significantly improved the delayed bactericidal activity against Staphylococcus aureus. Both intraperitoneal and oral administration of E-64-d to beige mice resulted in a significant increase in survival, suggesting that the administration of E-64-d may be effective against severe bacterial infection in Chediak-Higashi syndrome 7.

Reduced resorption lacunae formation on dentine slices: The effects of specific and potent inhibitors of vacular-type H+-ATPase and a lysosomal cysteine proteinase, cathepsins, on ultrastructure, expression of these enzymes and resorptive function of cultured osteoclasts was analysed. E-64 showed no effect on demineralization of dentine slices, but markedly reduced resorption lacunae formation on dentine slices, but had no effect on decalcification of apatite crystals. Results suggest that E-64 treatment decreases the depths of resorption lacunae in a dose-dependent manner, but by inhibition of secreted cathepsin K activity, and not by impairment of the ruffled border formation and associated the expression of vacular-type H+-ATPase and cathepsin K in osteoclasts .

References

1.     Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada K (1982). 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., 201:189-198.

2.     Hanada K, Tamai M, Yamagishi M, Ohmura S, Sawada J, Tanaka I (1978). Isolation and characterization of E-64, a new thiol protease inhibitor. Agri. Biol Chem., 42:523-528.

3.     Powers JC, Asgian JL, Ekici OD, James KE (2002). Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem Rev., 102(12):4639-4750.

4.     Hanada K, Tamai M, Morimoto S, Adachi T, Ohmura S, Sawada J, Tanaka I (1978). Inhibitory activities of E64derivatives of papain. Agric. Biol. Chem., 42:537-541.

5.     Sugita H, Ishiura S, Suzuki K, Imahori K (1980). Inhibition of epoxide derivatives on chicken calcium-activated neutral protease (CANP) in vitro and in vivo. J. Biochem., 87:339-341.

6.     Towatari T, Tanaka K, Yoshikawa D, Katunuma N (1978). Purification and properties of a new cathepsin from rat liver. J. Biochem., 84(3):659-671.

7.     Michiko M, Fuminori T, Hirotake K, Masahiko I (2007). Effect of a thiol proteinase inhibitor, E-64-d, on susceptibility to infection with Staphylococcus aureus in Chediak-Higashi syndrome (beige) mice. International immunopharmacology, 7(7):973-980.

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