Recombinant soluble TMPRSS2 ectodomain was purified and activated for peptidase activity inhibition assays as described previously . A total of 73 analogs were tested for TMPRSS2 inhibition using the commercial fluorogenic peptide substrate Boc-QAR-AMC (Extended Data Figs , Extended Data Tables ). For Scaffold 1, the addition of a methyl group in the para-position provided the best improvement in IC50 to 36 nM ('8). Other aryl and alkyl substitutions at para-and meta-resulted in Scaffold 1 compounds with IC50s ranging from 52 to 154 nM (Extended Data Fig. ; Extended Data Table ). Modifications to the Scaffold 1 ortho position ('6 and '3) produced weaker, micromolar IC50 inhibitors. The ester reversal of Scaffold 2 that placed the alpha-amino ketone on the benzoate phenyl in also resulted in low micromolar IC50 inhibitors (Extended Data Fig. ; Extended Data Table ). Scaffolds 3 and 4 yielded 16 compounds with nanomolar IC50 values by using amino methyl or amino ethyl S1-targeting head groups, respectively (Fig. ). Interestingly, Scaffold 5 compounds that were analogs of Scaffold 4 compounds did not substantially change the TMPRSS2 IC50 despite the replacement of the Scaffold 4 primary amine with a 3oxyazetdine. Incorporation of chemical moieties from nafamostat decreased IC50 values to single digit nanomolar for Scaffold 6 inhibitors with the introduction of the guanidinium group, and Scaffolds 3 and 4 with the addition of napthyl-amidine. This trend did not hold true for the incorporation of napthyl-amidine for Scaffold 5 resulting in an inhibitor with an IC50 greater than 4 µM ('158; Extended Data Table ). Based on these data, 23 compounds (Fig. ) were prioritized for subsequent biochemical and biophysical characterization.