ABSTRACT
The main protease (Mpro) of SARS-CoV-2, a cysteine protease that plays a key role in generating the active proteins essential for coronavirus replication, is a validated drug target for treating COVID-19. The structure of Mpro has been elucidated by macromolecular crystallography, but owing to its conformational flexibility, finding effective inhibitory ligands was challenging. Screening libraries of ligands as part of EXaSCale smArt pLatform Against paThogEns (ExScalate4CoV) yielded several potential drug molecules that inhibit SARS-CoV-2 replication in vitro. We solved the crystal structures of Mpro in complex with repurposed drugs like myricetin, a natural flavonoid, and MG-132, a synthetic peptide aldehyde. We found that both inhibitors covalently bind the catalytic cysteine. Notably, myricetin has an unexpected binding mode, showing an inverted orientation with respect to that of the flavonoid baicalein. Moreover, the crystallographic model validates the docking pose suggested by molecular dynamics experiments. The mechanism of MG-132 activity against SARS-CoV-2 Mpro was elucidated by comparison of apo and inhibitor-bound crystals, showing that regardless of the redox state of the environment and the crystalline symmetry, this inhibitor binds covalently to Cys145 with a well-preserved binding pose that extends along the whole substrate binding site. MG-132 also fits well into the catalytic pocket of human cathepsin L, as shown by computational docking, suggesting that it might represent a good start to developing dual-targeting drugs against COVID-19. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.