FIG 5.
Telaprevir binds EV-D68 2Apro in a biphasic irreversible reaction. (A) Proteolytic progress curves in the absence or presence of various concentrations of telaprevir. The EV-D68 2Apro protein (0.2 μM) was added to 80 μM FRET-1 substrate with various concentrations of telaprevir in 200 μl of reaction buffer at 30°C to initiate the proteolytic reaction. The reaction was monitored for over 6 h. The details of the curve fitting are detailed in Materials and Methods. E, enzyme; I, inhibitor. (B) Drug release dialysis of the 2Apro-telaprevir complex. EV-D68 2Apro (1.0 μM) was treated with 2.0 μM telaprevir or DMSO for 1 h at 30°C before dialyzing at 4°C. Aliquots were taken every 24 h to measure activity (the values are the mean ± SE from two replicates). Red asterisks indicate that no protease activity was detected. (C) The best-fit Kobs values in panel A were replotted against the inhibitor concentration with the equation Kobs = k2[I]/(KI + [I]), where the best-fit k2 was equal to 0.0011 ± 0.0003 s−1, KI was equal to 0.225 ± 0.063 μM (n = 3), and the calculated k2/KI was 4,750 M−1 s−1.