Extended Data Fig. 4: Recombinant S protein is differentially cleaved by dasTMPRSS2 and furin and is a useful substrate for dasTMPRSS2 inhibitor characterization.
From: Structure and activity of human TMPRSS2 protease implicated in SARS-CoV-2 activation
a Recombinant furin (NEB enzyme units indicated) cannot cut SARS-CoV-2 S protein with a KO at the S1/S2 cleavage site (HexaPro), but can cut S1/S2 intact S protein at S1/S2 over a period of 16 hrs at room temperature. 4 µg protein were loaded per well and visualized with Coomassie blue and gel results were consistent across n = 3 independent biological experiments. b HexaPro S protein digestion across 120 min with 300 nM dasTMPRSS2 following 15 min pre-incubation with 100:1 to 0.00001:1 nafamostat:dasTMPRSS2. 4 µg protein were loaded per well and visualized with Coomassie blue and gel results were consistent across n = 3 independent biological experiments. c Attempted IC50 plot of bromhexine (with its chemical structure shown) of residual dasTMPRSS2 peptidase activity in the presence of 10 µM Boc-QAR-AMC substrate showing no detectable inhibition. Data are shown as mean ± SD for n = 3 technical replicates. d Pre-incubation of bromhexine with 300 nM TMPRSS2 for 15 min results in no inhibition of proteolytic activity towards SARS-CoV-2 S protein construct HexaPro digested over 30 minutes. 4 µg protein were loaded per well and visualized with Coomassie blue and gel results were consistent across n = 3 independent biological experiments.
