In vitro activity: 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.

Kinase Assay: The Cathepsin B 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.

Cell Assay: E-64 inhibited H-59 invasion in a dose-dependent manner with a maximal inhibition of 97% at a concentration of 10 μg/ml which 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.