Covalent DNA-Encoded Library Workflow Drives Discovery of SARS-CoV-2 Nonstructural Protein Inhibitors

The COVID-19 pandemic, exacerbated by persistent viral mutations, underscored the urgent need for diverse inhibitors targeting multiple viral proteins. In this study, we utilized covalent DNA-encoded libraries to discover innovative triazine-based covalent inhibitors for the 3-chymotrypsin-like protease (3CL^pro, Nsp5) and the papain-like protease (PL^pro) domains of Nsp3, as well as novel non-nucleoside covalent inhibitors for the nonstructural protein 12 (Nsp12, RdRp). Optimization through molecular docking and medicinal chemistry led to the development of LU9, a nonpeptide 3CL^pro inhibitor with an IC₅₀ of 0.34 μM, and LU10, whose crystal structure showed a distinct binding mode within the 3CL^pro active site. The X-ray cocrystal structure of SARS-CoV-2 PL^pro in complex with XD5 uncovered a previously unexplored binding site adjacent to the catalytic pocket. Additionally, a non-nucleoside covalent Nsp12 inhibitor XJ5 achieved a potency of 0.12 μM following comprehensive structure–activity relationship analysis and optimization. Molecular dynamics revealed a potential binding mode. These compounds offer valuable chemical probes for target validation and represent promising candidates for the development of SARS-CoV-2 antiviral therapies.