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Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an MProsubstrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MProcatalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MProinhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50value in inhibiting SARS-CoV-2 from infecting ACE2+A549 cells and a selectivity index of 875. The crystallography analyses of MProbound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MProinhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MProas therapeutics for COVID-19.

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (MPro) for replication and pathogenesis. MProis a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MProinhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 reversibly covalent dipeptidyl MProinhibitors and characterized them onin vitroenzymatic inhibition potency, structures of their complexes with MPro, cellular MProinhibition potency, antiviral potency, cytotoxicity, andin vitrometabolic stability. Our results indicated that MProhas a flexible S2 pocket that accommodates dipeptidyl inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro[4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have optimal characteristics. One compound MPI60 containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and highin vitrometabolic stability and can be potentially advanced to further preclinical tests.

As an essential enzyme to SARS-CoV-2, main protease (MPro) 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 MPro 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 MPro 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 MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro 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 MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.