E-64 (Proteinase inhibitor E 64) is an irreversible and specific cysteine protease inhibitor. The IC50 of E-64 for papain is 9 nM.

Cysteine protease:9 nM

E-64迅速抑制半胱氨酸蛋白酶如cathepsins B、H、L以及papain的活性，但对丝氨酸蛋白酶或金属蛋白酶无影响。[2]作为特异性半胱氨酸蛋白酶抑制剂，E-64有效阻止体外卵巢癌细胞侵袭。[3]此外，E-64还展现了针对体外Giardia lamblia excystation的抗寄生虫活性。[4]E-64改善牛体细胞核移植胚胎的种植前发展，这表明了E-64的另一个重要作用。[5]

E-64显著减少了携带M5076肿瘤的小鼠中自发性转移的数量，但对实验性转移的形成无影响。[6] E-64还对感染小鼠中的贾第鞭毛虫(excystation)表现出抗寄生虫活性。[4]

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[2].