Optimization of SARS-CoV-2 M^pro Inhibitors by a Structure-Based Multilevel Virtual Screening Method

With the aim of developing novel anti-SARS-CoV-2 drugs to address the ongoing evolution and emergence of drug-resistant strains, the reported SARS-CoV-2 M^pro inhibitor WU-04 was selected as a lead to find novel, highly potent, and broad-spectrum inhibitors. Using a fragment-based multilevel virtual screening strategy, 15 hit compounds were identified and subsequently synthesized. Among them, A5 (IC₅₀ = 1.05 μM), A6 (IC₅₀ = 1.08 μM), and A9 (IC₅₀ = 0.154 μM) demonstrated potent SARS-CoV-2 M^pro inhibition comparable to or slightly weaker than WU-04. Antiviral activity evaluations revealed that compound A9 exhibited the strongest antiviral activity with an EC₅₀ value of 0.18 μM, quite comparable to the marketed drug Nirmatrelvir (EC₅₀ = 0.123 μM) and inferior to WU-04 (EC₅₀ = 0.042 μM). Molecular dynamics simulations elucidated the key interactions between compounds A5, A6, A9, and the binding pocket of SARS-CoV-2 M^pro, providing valuable insights into their mechanisms of action. These findings identify compound A9 as a promising lead for anti-SARS-CoV-2 drug development.