Biochemical analyses of X77, S-X77, Rac-X77 The SARS-CoV-2 Mpro was synthesized using the ORF1ab polyprotein residues 3264-3569, (GenBank code: MN908947.3). Gene synthesis, protein production, and purification were as reported by Zhang et al., where eluted fractions containing the target protein were pooled and subjected to buffer exchange in 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 7.8. The detection of enzymatic activity of the Mpro was performed under conditions reported by Kuzikov et al. . Enzymatic activity was measured by a Förster resonance energy transfer (FRET), using the dual-labeled substrate, DABCYL-KTSAVLQ↓SGFRKM-EDANS (Bachem #4045664) containing a protease specific cleavage site after the GLN. In the intact peptide, EDANS fluorescence is quenched by the DABCYL group. Following enzymatic cleavage, generation of the fluorescent product was monitored (Ex/Em = 340/460 nm) (EnVision, PerkinElmer). The assay buffer contained 20 mM Tris (pH 7.3), 100 mM NaCl, and 1 mM EDTA. The assay was established in an automated screening format (384 well black microplates, Corning, #3820) and optimized with respect to assay volume (10 μL), enzyme concentration (60 nM), substrate concentration (15 μM), incubation time (60 min with compounds, 15 min with substrate) and temperature (37 °C for incubation with compounds, 25 °C for incubation with substrate), DMSO tolerance (up to 5 v/v%), response to inhibition with known compounds as zinc pyrithione and the effects of reducing agents (DTT). X77, S-X77 and Rac-X77 were then profiled in triplicate in 11 points concentration responses, starting from 20 μM top concentration with 1:2 dilution steps.