FIG 6.
T26I/D65G MHV 3CLpro has reduced enzymatic efficiency and is not efficiently blocked by GRL-001. The enzymatic efficiency (kapp) (A) and inhibition by GRL-001 (IC50) (B) of both WT and T26I/D65G 3CLpro were determined at ambient temperature (25°C) and 37°C. (A) The initial reaction rates (Vi) were determined by calculating the initial slope of the progress curve, which was then converted to the amount of product (μM) produced per minute using the experimentally determined value of the fluorescence extinction coefficient for UIVT3. Plotting Vi/[E] versus [UIVT3] gave a linear correlation, the slope of which was taken to be the kapp. (B) The inhibition of the 3CLpro by GRL-001 was monitored by following the change in RFUs over time, using the initial slope of the progress curve to determine the initial rate. The percent inhibition of the 3CLpro enzymes was then plotted as a function of inhibitor concentration. (C) kapp and IC50s were determined using the nonlinear regression program SigmaPlot.