To accurately rank the esters based on their covalent reactivity and binding potency, TMPRSS2 acylation kinetic studies were performed with the identified hits and compared to known covalent inhibitors nafamostat, camostat, and 6-amidino-2-naphthol. In this kinetic assay, compound inhibition was interrogated against 1.6 nM recombinant TMPRSS2 (Fig. ). Real-time acylation of the TMPRSS2 active site was measured by co-addition of substrate and inhibitor to microplates containing enzyme, as performed previously for TMPRSS2 with nafamostat and camostat . A subset of the covalent inhibitor series was tested, including '157, '156, '8, and '148. Esters showed a plateau of enzyme activity over time consistent with the formation of an inactive acyl-enzyme complex (Fig. ; Extended Data Fig. ). The kinact/KI values of '157 and '156 were 0.93, and 1.6 µM -1 s -1 , respectively, and were comparable to nafamostat's kinact/KI value of 2.8 µM -1 s -1 (Fig. ; Extended Data Fig. ). However, amongst these potent ester inhibitors, only the TMPRSS2 reaction progress curves in the presence of nafamostat showed a complete plateau of TMPRSS2 peptidase activity, indicating the 4-guanidino benzoate acyl-enzyme complex was stable and did not rapidly hydrolyze across the analyzed reaction time. Less potent inhibitors '8 and '148 had kinact/KI values of 0.0045 and 0.0036 µM -1 s -1 , respectively, which were comparable to camostat's kinact/KI value of 0.085 µM -1 s -1 (Extended Data Fig. ). In contrast, noncovalent inhibitors 6amidino-2-naphthol, '2222 and '2602 showed dose-dependent depression of enzyme activity, but no plateau of enzymatic activity over time (Fig. ). Accordingly, TMPRSS2 Ki values of 1.1, 13, and 40 µM were determined for 6-amidino-2-naphthol, '2602, and '2222, respectively.