Fig. 5: Probing PPACK-Mpro interaction by enzyme inhibition assays.
A Bar plot of Mpro inhibition by argatroban and PPACK. Argatroban or PPACK (7 μM) was incubated for 2 h in HBS-PEG, pH 7.4, at 37 ± 0.1 °C with rcMpro (50 nM). The reaction was started by the addition of the fluorogenic substrate (1.25 μM), and the rate of hydrolysis was determined by recording the fluorescence increase at 530 nm. The data are expressed as the relative rate (vi/v0) of substrate hydrolysis in the presence (vi) and absence (v0) of the inhibitor (n = 2). B Determination of Mpro inhibition constant by PPACK. Enzyme inhibition assays were carried out as in (A). The values of (vi/v0) are plotted as a function of inhibitor concentration. The data points were interpolated with Eq. 2, describing the tight binding inhibition model, to extract the apparent inhibition constant (Kiapp), from which a value of Ki = 160 ± 2 nM was estimated (see “Methods”) (n = 1).
