Covalent inhibitors are small molecules that form covalent bonds with nucleophilic residues such as cysteines in target proteins. Their unique interactions offer advantages such as enhanced potency, prolonged action, and high selectivity, particularly when targeting unique binding pockets or reactive residues. Covalent inhibitors have been applied in various therapeutic targets in drug development. 

Traditionally, high-throughput screening of covalent inhibitors relies on commercial covalent fragment libraries and activity-based protein profiling. In a study published in Journal of the American Chemical Society, a team led by Prof. LU Xiaojie and Prof. XU Yechun from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences introduced a novel covalent DNA-encoded library (DEL) workflow which enables covalent screening against cysteine-rich non-structural proteins of SARS-CoV-2, leading to the identification of covalent hit compounds with novel scaffolds and allosteric binding modes.

In this study, researchers focused on SARS-CoV-2 non-structural proteins. Structural analyses identified three key targets: Nsp3 (PL^pro), Nsp5 (3CL^pro), and the cysteine-rich Nsp7/8/12 complex. The covalent DEL workflow was specifically designed by introducing a covalent linker as the final building block during library synthesis, enabling selective and rational design to target deeply buried reactive residues. Using wild-type and catalytically inactive (Cys-mutant) protein variants, comparative screenings were conducted to identify covalent inhibitors targeting catalytic residues. 

For the first time, researchers discovered compounds binding at an allosteric site outside the ubiquitin pocket of PL^pro. By correlating IC₅₀ values with standardized enrichment, they predicted activity across diverse scaffolds. For 3CL^pro, researchers achieved precise docking by introducing conformational constraints to DNA-linked compounds exposed to solvent regions, and this was further validated by protein-ligand crystal structures. Moreover, covalent analysis of Nsp7/8/12 via gel electrophoresis and quantum mechanics/molecular mechanics simulations revealed the potential binding sites.

This study offers insights into the structural basis of viral protease inhibition, and lays the groundwork for rational drug design and optimization.