Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19. [Display omitted] •Azapeptides are a viable scaffold for the design of SARS-CoV-2 main protease inhibitors.•Diverse covalent warheads can be readily attached to azapeptides.•MPI89 with an aza-2,2-dichloroacetyl warhead has an EC50 value of 10 nM against SARS-CoV-2.•Crystallography analysis indicates most inhibitors form a covalent bound with the MPro active site cysteine.•MPI89 maintains high potency against the E166V/L50F mutant of MPro.