The P3 O-Tert-Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors

As an essential enzyme to SARS-CoV-2, main protease (M^Pro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of M^Pro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC₅₀ values that range from 4.8 to 650 nM. The determined IC₅₀ values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro M^Pro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of M^Pro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the M^Pro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In M^Pro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of M^Pro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of M^Pro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.