Fig. 3: Nafamostat rapidly acylates dasTMPRSS11D, then hydrolyzes to restore protease activity.
From: Structural basis of TMPRSS11D specificity and autocleavage activation
a The putative nafamostat covalent inhibition mechanism for TMPRSS11D. b Peptidase activity progress curves of dasTMPRSS11D (3 nM) with nafamostat at the indicated inhibitor concentrations added simultaneously with Boc-QAR-AMC substrate (100 µM final). c Peptidase activity progress curves of dasTMPRSS11D (8 nM) pre-incubated (10 min) with the indicated concentrations of nafamostat before being transferred to wells containing Boc-QAR-AMC substrate (100 µM final). Data for (b, c) are shown as mean values for experiments performed in technical duplicate (n = 2) and are consistent with results obtained across n = 4 independent biological replicates d Melting temperature shifts (ΔTms) of dasTMPRSS11D protein in the presence of the indicated concentrations of nafamostat (teal datapoints) or 6-amidino-2-naphthol (violet datapoints) ligands. Each assay contained 2 µg dasTMPRSS11D, 5X SYPRO orange dye, and 50 mM Tris pH 8.0 with 200 mM NaCl. Data are shown as mean values for experiments performed in technical triplicate (n = 3), with consistent data observed across n = 3 independent biological replicates. The ΔTm data were curve-fitted for one-site EC50 in GraphPad Prism.
