Protease cleavage tests were done for the compound sets of Nsp5, Nsp3, and TMPRSS2. In the assay, a peptide FRET (Förster resonance energy transfer) substrate is cleaved by the protease, which results in an increase of fluorescence intensity. The increase in fluorescence intensity over time is proportional to the rate constant of the protease, and by adding compounds at different concentrations, inhibitors can be identified. As positive inhibitor controls, GC376 (IC 50 = 9.4 ± 2.5 nM) and GRL0617 (IC 50 = 2.8 ±0.4 µM) were used for Nsp5 and Nsp3, respectively (see Supporting Information Section 4). A first brute-force screening at 100 µM showed a single compound for each of the three proteases (see red bars in Figure ). Those compounds were selected for dose-response curves, where their concentration was changed to calculate IC 50 values (see Supporting Information Section 4). Nsp5-1 produced an atypical dose-response, where activity was first enhanced by ~50% and then dropped to < 50% at 100 µM concentration (Figure ), which hampered the calculation of the IC 50 . Nsp3-1 showed a classical inhibition with IC 50 = 24.7 ± 3.7 µM (Figure ). In addition, from cell-based Nsp5 assays (see section 2.6.2 below), 5 additional compounds were identified that did not make the < 50% inhibition threshold, but were measured in dose-response using the same cleavage assay (Figure ). These measurements identified the IC 50 of Nsp5-2 ~288 µM, whereas the remaining compounds Nsp5-3 to Nsp5-6 had much higher IC 50 's that could not be determined.