Detection of inhibitors by fluorescence spectrophotometry. For all inhibition experiments, reactions were performed in 100 µL assay with 0.1 or 10 µM of the fluorescence substrates ubiquitin or Z-RLRGG-AMC, respectively from BPS Biosciences (San Diego, CA, USA), which has been previously used for assaying PL pro proteases. 10 µL of 0.8 µM enzyme were mixed with 10 µL of the inhibitor and 70 µL of assay buffer (50 mM Tris-HCl, 150 mM NaCl, 1.5 mM DTT, 0.1 mg/ml BSA) for 30 mins at room temperature. In both screening (duplicates) and IC50 experiments (duplicates or triplicates), 10 µL of 1 or 30 µM of the fluorescence substrates ubiquitin or Z-RLRGG-AMC, respectively diluted in assay buffer with were added and the reactions were monitored by following the fluorescence as a function of time (excitation at 360 nm, emission at 460 nm) using a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (Winooski, VT, USA). Controls were (i) positive control, 2 µL of DMSO, (ii) inhibitor control, 2 µL of 500 µM GRL0617 (synthetized by us) (iii) blank, the PL pro without inhibitor. For screening, the compounds were added as DMSO solutions and their final concentration in the reactions was 50 µM. For IC50 measurement, serial dilutions were performed to reach a final concentration ranging from 200 µM to 0.2 µM. The reactions ran for at least one hour, and the linear initial slopes of the progress curves were used to calculate the reaction initial velocity in Relative Fluorescent Units in time (RFU per min). GraphPad software was used to determine IC50 values.