A homology model of TMPRSS2 was initially constructed, since at the onset of this study, the crystal structure of TMPRSS2 had not been published. The homology model was constructed from plasma kallikrein, coagulation factor XI, TMPRSS11E, matriptase, and Coagulation factor VII and exclusively spanned the TMPRSS2 serine protease domain (Fig. ; Extended Data Fig. ). The model was found to be highly similar to the experimentally determined crystal structure of TMPRSS2 following its acylation by nafamostat (Fig. ). The TMPRSS2 homology model was docked against the ZINC20 library 42 , which includes approximately 200 million monocationic compounds, primarily sourced from the make-ondemand Enamine REAL database (Extended Data Fig. ). Of the compounds docked within the TMPRSS2 active site, 121 were initially selected for recombinant TMPRSS2 inhibition screening and a single hit ('2805) was obtained (0.8% hit rate; Extended Data Fig. ) with a phenyl benzoate ester and a primary amino group. The nafamostat and camostat TMPRSS2 inhibition mechanism involves the formation of an inactive acyl-enzyme complex (Fig. ), and since '2805 also contains an ester group, we hypothesized that '2805 may form an inactive acyl-enzyme complex (Fig. ). Using biochemical TMPRSS2 peptidase inhibition assays, '2805 was found to have a TMPRSS2 half-maximal inhibitory concentration (IC50) of 120 nM (Fig. ). As shown in the '2805:TMPRSS2 docked pose, the primary amine in '2805 likely binds the TMPRSS2 S1, similar to the guanidinium found in nafamostat and camostat. The ester linkage in '2805 is reversed with respect to the orientation of the cationic groups found in nafamostat and camostat, however, the '2805 ester group is close in proximity to the TMPRSS2 S441 residue to enable nucleophilic attack and acyl-enzyme complex formation (S441*; Fig. ).