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

The main protease (M^Pro) 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 M^Pro's catalytic cysteine. Our characterization identified potent M^Pro 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 M^Pro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.