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E-64 is a natural, potent and irreversible inhibitor of cysteine proteases.1 Its IC50 values for inhibiting cathepsins K, S, and L, in vitro, are 1.4, 4.1, and 2.5 nM, respectively.2 E-64 also inhibits papain, calpain, and cathepsins B and H, but not serine proteases (e.g., plasmin, trypsin, tissue kallikrein) or aspartic proteases (e.g., pepsin).3 The mode of action involves the formation of a thioether linkage between E-64 and the thiol group of the protease; E-64 does not react with the functional thiol group of non-protease enzymes (e.g., creatine kinase, L-lactate hydrogenase). While E-64 is usually used in biochemical assays, it can also be used in intact cells at higher (μM) concentrations.4,5,6
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1
Drahl, C., Cravatt, B.F., Sorensen, E.J. Protein-reactive natural products. Natural Products Chemistry 44 5788-5809 (2005).
2
Susa, M., Luong-Nguyen, N., Cappellen, D., et al. Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay. Journal of Translational Medicine (2004).
3
Barrett, A.J., Kembhavi, A.A., Brown, M.A., et al. 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 (1982).
4
Giusti, I., D'Ascenzo, S., Millimaggi, D., et al. Cathepsin B mediates the pH-dependent proinvasive activity of tumor-shed microvesicles. Neoplasia 10(5) 481-488 (2008).
5
Ahlberg, J., Berkenstam, A., Henell, F., et al. Degradation of short and long lived proteins in isolated rat liver lysosomes; effects of pH, temperature, and proteolytic inhibitors. J Biol Chem 260(9) 5847-5854 (1985).
6
Drew, M.E., Banerjee, R., Uffman, E.W., et al. Plasmodium food vacuole plasmepsins are activated by falcipains. J Biol Chem 283(19) 12870-12876 (2008).
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