Our first series of compounds was identified through a large library docking campaign against a homology model of TMPRSS2, before the first TMPRSS2 crystal structure became available. Over 200 million compounds were virtually screened , identifying '2805 which was predicted to bind within the S1 of TMPRSS2 and had a reactive ester group. Our biochemical data confirmed that '2805 potently inhibited TMPRSS2 protease activity and likely formed an acyl-enzyme complex with TMPRSS2 following nucleophilic attack on '2805's ester using TMPRSS2's S441 residue. To improve TMPRSS2 binding affinity, we installed various modifications to '2805, producing 6 distinct ester inhibitor scaffolds. We employed a combination of kinetic TMPRSS2 inhibition assays and DSF ligand binding assays to interrogate noncovalent binding affinity and covalent reactivity of the ester scaffolds. We compared the inhibition potencies of these esters to nafamostat, a potent TMPRSS2 inhibitor that had been used in exploratory clinical trials for anti-TMPRSS2 COVID-19 therapy. The most potent TMPRSS2 inhibitors were '156 and '157 which contained the same naphthyl amidine as nafamostat. We determined the structure of the TMPRSS2 following incubation with '157 which confirmed that a 4-ethyl amino benzoate acyl-enzyme complex was formed. However, to understand the potency of these compounds, we solved the crystal structure of TMPRSS2 bound to 6-amidino-2-naphthol and showed that these compounds could potentially bind the TMPRSS2 S1 using their amidines or their S1-targeting headgroups before TMPRSS2 nucleophilically attacks their esters. To provide evidence for this amidine-mediated noncovalent binding for nafamostat, '157, and '156, we showed that these compounds could induce Tm shifts in the TMPRSS2 S441A protein, similar to 6-amidino-2-naphthol. In contrast, camostat, which lacks the napthyl amidine, could not induce Tm shifts for the TMPRSS2 S441A protein. These data explained how nafamostat more potently disables TMPRSS2 activity (and other trypsin-like serine proteases) than other related esters containing a single cationic, S1-targeting head group. However, both nafamostat and '157 were found to potently disable the activity of various trypsin-like serine proteases (Extended Data Fig. ), indicating that future chemical modifications to the ester scaffold may be necessary to confer TMPRSS2 selectivity.