Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. The SARS-CoV-2 (SC2) genome encodes for 10 open reading frames (ORFs), of which ORF1ab is the largest one that encodes two polyproteins pp1ab and pp1a. The two polyproteins are cleaved by two proteases, namely main protease (Mpro), and papain-like protease (PLpro) to yield 16 non-structural proteins (nsps). Mpro operates at 11 cleavage sites on the large polyprotein pp1ab to process 12 out of the total 16 nsps, which play critical role in viral replication and pathogenesis. Therefore, inhibition of this protease is sufficient in preventing the virus from replicating in infected cells which makes it a potential drug target. 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. Scrutinizing the substrate preference of SC2Mpro and inspired by the previous works on the development of inhibitors against SC1Mpro, the Liu lab has developed reversible covalent inhibitors to target SC2Mpro. By bringing about structural manipulation to the amino acid residues in the inhibitor design to improve cell membrane permeability and metabolic stability, we have generated a cohort of peptidomimetic inhibitors that target SC2Mpro.
In a different approach, by converting the P1 Ca atom in an M^Pro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the M^Pro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent M^Pro 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 2,000. The crystallography analyses of M^Pro 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 M^Pro 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 M^Pro as therapeutics for COVID-19.

Shiqing Xu

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