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 (3CLpro, Nsp5) and the papain-like
protease (PLpro) 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 3CLpro inhibitor with an IC50 of 0.34 μM, and LU10, whose crystal structure showed a distinct binding mode within the
3CLpro active site. The X-ray cocrystal structure of SARS-CoV-2
PLpro 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.