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. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MPro inhibitors. 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 MPro inhibitors and characterized them on in vitro enzymatic inhibition potency, structures of their complexes with MPro, cellular MPro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that MPro has 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 high in vitro metabolic stability and can be potentially advanced to further preclinical tests.

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 C(alpha) atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound 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 MPro inhibitors 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 MPro as therapeutics for COVID-19.