in Chart 1, which forms a covalent bond with CYS145, and its IC50 for MPro is 7.4 μM . The MX structure bound to MG-132 with double occupancy was solved previously by some of us at 1.94 Å in the C2 space group (PDBid 7NF5) and also, at 1.68 Å resolution in the P212121 space group (PDBid 7BE7), in condition of excess of ligands The second ligand is the R-enantiomer N-(4-tert-butylphenyl)-N-[(1R)-2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl]-1H-imidazole-4-carboxamide, X77 in Chart 1, which forms only non covalent interactions. The MX structure with double ligand occupation was reported at 2.1 Å in the C2 space group (PDBid: 6W63) and we reproduced it in our crystallization condition in spacegroup P212121 (PDBid: 7PHZ). Its inhibitory activity for Mpro, along with that of the S-enantiomer (S-X77 hereafter) and that of the racemate (rac-X77), were not known when we started this study. They were measured here employing a Förster resonance energy transfer (FRET) with a duallabeled 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. Its inhibitory activities are reported in Fig. as dose-response curves. X77 and S-X77 were identified by a comparison with Xray experiments, where the two enantiomers were separately co-crystallized with Mpro, solving 4 crystal structures, with the two enantiomers at two different concentrations (see below paragraph). The inhibitory activities are reported in Fig. as dose-response curves. The racemate showed an IC50 of 3.7 µM, while that of X77 is 1.7 µM. The S-X77 curve could not allow IC50 calculation as no real dose-response could be measured: likely, this enantiomer could not properly bind to stop the reaction. Indeed, this was confirmed by solving the crystal structure with the S-X77 enantiomer (see below paragraph).