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

High Resolution Image Download MS PowerPoint Slide 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 IC 50 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 N Ar) 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 IC 50 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.