High-Throughput Screening Identifies Covalent Inhibitors with Unique Chemistry and Binding Modes for SARS-CoV‑2 Main...

Using an easily scalable SARS-CoV-2 main protease (M^Pro) substrate, high-throughput screening of ∼75,000 compounds drawn from diversity and fragment libraries was conducted. Based on screening results, 40 inhibitors were confirmed with IC₅₀ values below 30 μM. Four hits were revalidated, and their M^Pro complexes were characterized by X-ray crystallography. For two inhibitors, CDI5573 and CDI8009, clear active-site electron density revealed covalent modification of Cys145, the enzyme's catalytic cysteine. In both structures, His41 of the catalytic dyad reorients from its native hydrogen-bonding pose with Cys145 to engage in π-stacking with the inhibitors' aromatic rings. The two covalent adducts arise from distinct chemical mechanisms. CDI5573, which is a 3-nitro-1,2,5-oxazole derivative, reacts through nucleophilic aromatic substitution (S_NAr) to replace one of the nitro groups by Cys145, whereas CDI8009 undergoes nucleophilic acyl substitution to form a thioester with Cys145. Structure-guided optimization of CDI5573 yielded new analogs, including one inhibitor with an IC₅₀ of 15 nM, placing this molecule among the most potent inhibitors developed for M^Pro. These findings introduce new covalent chemotypes with diverse modes of engagement for M^Pro, expand the accessible space for this essential viral enzyme, provide leads with the potential to complement existing COVID-19 therapeutics and mitigate viral resistance, and guide the targeting of other cysteine enzymes for drug discovery.