Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen

Kuzikov, Maria; Costanzi, Elisa; Reinshagen, Jeanette; Esposito, Francesca; Vangeel, Laura; Wolf, Markus; Ellinger, Bernhard; Claussen, Carsten; Geisslinger, Gerd; Corona, Angela; Iaconis, Daniela; Talarico, Carmine; Manelfi, Candida; Cannalire, Rolando; Rossetti, Giulia; Gossen, Jonas; Albani, Simone; Musiani, Francesco; Herzog, Katja; Ye, Yang; Giabbai, Barbara; Demitri, Nicola; Jochmans, Dirk; Jonghe, Steven De; Rymenants, Jasper; Summa, Vincenzo; Tramontano, Enzo; Beccari, Andrea R.; Leyssen, Pieter; Storici, Paola; Neyts, Johan; Gribbon, Philip; Zaliani, Andrea

[Image: see text] Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC(50) values below 1 μM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.