Compounds that reduced TMPRSS2 peptidase activity by ≥ 40% were characterized in dose-response experiments. Assays were run on a BioTek Synergy H4 Plate Reader using 384-well plates. Compounds were plated in a 96 well plate (clear, round bottom) in buffer (180 µL, 50 mM Tris, 150 mM NaCl, 0.02% TWEEN-20, pH= 8) at various concentrations. TMPRSS2 protein was thawed on ice and diluted into a buffer (3 mL buffer, 420 pM (3x)). The enzyme was first plated (7 µL, final concentration 140 pM) followed by compound (7 µL, final concentration 100 µM). The enzyme and compounds were incubated 20 min at RT followed by 5 min at 37ºC. After incubation, substrate Boc-QAR-AMC (7 µL, final concentration, 133 µM, ex: 340 (20), em: 441(20)). Fluorescent emissions were read every 1 to 5 mins over 2 hours. Wells containing substrate only (no inhibitor or enzyme) were treated as no enzyme controls (NEC). Wells containing enzyme and substrate (no inhibitor) were treated as positive controls. Average velocities were calculated for all wells (for points between 10 minutes and 30 minutes) using Gen5 software. Percent activity for all wells were calculated and plotted in GraphPad Prism. IC50 curve fittings were generated by GraphPad Prism non-linear fits using the "[Inhibitor] vs. response (three parameter)" function.