To test whether inhibitors blocked TMPRSS2 protease activity on a more physiological substrate, inhibition assays were performed using purified SARS-CoV-2 S protein as a substrate and reaction progress was monitored by SDS-PAGE and Coomassie blue staining (Fig. ). Inhibitors were pre-incubated for 30 min with TMPRSS2, then transferred to buffer containing the S protein substrate and relative S protein cleavage evaluated after 15 min. The inhibitory potencies of the ester compounds were similar to one another due to the long pre-incubation time with TMPRSS2 prior to the start of the assay, allowing complete acylation of the TMPRSS2 Ser441 residue to block activity (Fig. ). At low inhibitor concentrations, the esters did not show any detectable inhibition of dasTMPRSS2 protease activity. In contrast, the noncovalent inhibitors 6amidino-2-naphthol, '2222, and '2602 completely blocked TMPRSS2-mediated S protein cleavage at high inhibitor concentrations but showed partial TMPRSS2-mediated cleavage of the S protein as the inhibitor concentrations were lowered (Fig. ). These gel-based TMPRSS2 inhibition assays demonstrate that these compounds can disable TMPRSS2 activity towards SARS-CoV-2. The molecular docking screens predicted '2222 and '2602 were noncovalent TMPRSS2 inhibitors that form hydrogen bonds with the catalytic S441 residue. To experimentally test this prediction, we designed assays that could measure ligand binding in the presence or absence of the TMPRSS2 catalytic Ser441 residue.