Extended Data Fig. 2: Rapid nafamostat acylation of the dasTMPRSS2 catalytic serine is thermally stabilizing and slowly deacylates.
From: Structure and activity of human TMPRSS2 protease implicated in SARS-CoV-2 activation
a Apparent dasTMPRSS2 melting temperatures (as determined by differential scanning fluorimetry; Methods) are increased for benzamidine, camostat, and nafamostat at 1µM concentration but are not increased by SFTI-1 or bromhexine relative to control incubation with DMSO. Datapoints are shown as means ± SD. Samples were run in technical quadruplet and statistically significant differences (p<0.0001; denoted as ****) from control were determined by one-way ANOVA for n=4 biologically independent samples (or n=3 samples for SFTI-1 and benzamidine) examined over 1 independent experiment. b IC50 plots of camostat (left) and nafamostat (right) across increasing lengths of inhibitor pre- incubation time with dasTMPRSS2 enzyme, enabling calculation of kinact and Ki via Equations 2 and 3 (Methods). Datapoints are shown as means ± SD. Samples were run in technical duplicate (n=2) and curve fit for absolute IC50 in GraphPad. Consistent plots were obtained for n=3 independent biological experiments. c Fluorescence standard curve for the quantification of the nafamostat leaving group, 6-amidino-2-napthol. Samples were run in technical triplicate (n=3) and fit to a linear curve in GraphPad producing the indicated slope and R-squared values. d dasTMPRSS2 enzyme quantification after 5 min incubation with 1µM nafamostat and monitoring 6-amidino-2-napthol production as endpoint fluorescence (320:490). The indicated dilutions estimate a 3.2nM dasTMPRSS2 concentration with samples run in technical duplicate (n=2). e Residual dasTMPRSS2 peptidase activity monitored by turnover of Boc-QAR-AMC substrate following stoichiometric enzyme acylation by nafamostat. Enzyme co-incubations with nafamostat or DMSO were transferred to wells containing 250µM or 125µM substrate (final enzyme concentration of 3.2nM) and kinetic traces of nafamostat-treated samples were curve fit in GraphPad to calculate the half-life of activity recovery (Methods). f Chemical mechanism of hydrolysis of the phenylguanidino acyl-enzyme complex and recovery of dasTMPRSS2 peptidase observed kinetically in (e).
