SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Crystallization of Feline Coronavirus Mpro With GC376 Reveals Mechanism of Inhibition.

Coronaviruses infect a variety of hosts in the animal kingdom, and while each virus is taxonomically different, they all infect their host via the same mechanism. The coronavirus main protease (Mpro, also called 3CLpro), is an attractive target for drug development due to its essential role in mediating viral replication and transcription. An Mpro inhibitor, GC376, has been shown to treat feline infectious peritonitis (FIP), a fatal infection in cats caused by internal mutations in the feline enteric coronavirus (FECV). Recently, our lab demonstrated that the feline drug, GC373, and prodrug, GC376, are potent inhibitors of SARS-CoV-2 Mpro and solved the structures in complex with the drugs; however, no crystal structures of the FIP virus (FIPV) Mpro with the feline drugs have been published so far. Here, we present crystal structures of FIPV Mpro-GC373/GC376 complexes, revealing the inhibitors covalently bound to Cys144 in the active site, similar to SARS-CoV-2 Mpro. Additionally, GC376 has a higher affinity for FIPV Mpro with lower nanomolar Ki values compared to SARS-CoV and SARS-CoV-2 Mpro. We also show that improved derivatives of GC376 have higher potency for FIPV Mpro. Since GC373 and GC376 represent strong starting points for structure-guided drug design, determining the crystal structures of FIPV Mpro with these inhibitors are important steps in drug optimization and structure-based broad-spectrum antiviral drug discovery.