Covalent DNA-Encoded\nLibrary Workflow Drives Discovery\nof SARS-CoV‑2 Nonstructural Protein Inhibitors

The COVID-19 pandemic, exacerbated by persistent viral\nmutations,\nunderscored the urgent need for diverse inhibitors targeting multiple\nviral proteins. In this study, we utilized covalent DNA-encoded libraries\nto discover innovative triazine-based covalent inhibitors for the\n3-chymotrypsin-like protease (3CL<sup>pro</sup>, Nsp5) and the papain-like\nprotease (PL<sup>pro</sup>) domains of Nsp3, as well as novel non-nucleoside\ncovalent inhibitors for the nonstructural protein 12 (Nsp12, RdRp).\nOptimization through molecular docking and medicinal chemistry led\nto the development of <b>LU9</b>, a nonpeptide 3CL<sup>pro</sup> inhibitor with an IC<sub>50</sub> of 0.34 μM, and <b>LU10</b>, whose crystal structure showed a distinct binding mode within the\n3CL<sup>pro</sup> active site. The X-ray cocrystal structure of SARS-CoV-2\nPL<sup>pro</sup> in complex with <b>XD5</b> uncovered a previously\nunexplored binding site adjacent to the catalytic pocket. Additionally,\na non-nucleoside covalent Nsp12 inhibitor <b>XJ5</b> achieved\na potency of 0.12 μM following comprehensive structure–activity\nrelationship analysis and optimization. Molecular dynamics revealed\na potential binding mode. These compounds offer valuable chemical\nprobes for target validation and represent promising candidates for\nthe development of SARS-CoV-2 antiviral therapies.