FIGURE 9.

A, inhibition of ECE-2 enzymatic activity by phosphoramidon using either McabK₂ (▾) or PL405 (■) fluorogenic substrates. Increasing concentrations of phosphoramidon were incubated 10 min at 37 °C with 100 ng/ml of ECE-2 in 200 mm sodium acetate, pH 5.5. The reactions were initiated by the addition of either 10 μm McabK₂ or 20 μm PL405 followed by a 30- or 60-min incubation at 37 °C, respectively. B, inhibition of ECE-2 by various metalloprotease inhibitory compounds. Different concentrations of either compound 1 (■), kelatorphan (▴), retrothiorphan (▾), thiorphan (♦), or captopril (●) were incubated 10 min at 37 °C with 100 ng/ml of ECE-2 in 200 mm sodium acetate, pH 5.5. The reactions were initiated by the addition of 20 μm PL405 followed by a 60-min incubation at 37 °C. C, inhibitory potency of compound 2 against ECE-2 using the PL405 assay (■) compared with that against ECE-1 using the McabK₂ substrate (▴), revealing a discriminating factor of 62. The obtained K_i values from the conversion of the obtained IC₅₀ values are reported in Table 1.