Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

TLDR

Coronaviruses infect humans and animals, causing a spectrum of diseases, and their main protease is essential for viral replication, making it an attractive drug target. This study aimed to determine the crystal structures of IBV M pro and a SARS‑CoV M pro mutant H41A bound to an autocleavage substrate to elucidate structural flexibility and substrate binding. The authors crystallized the two proteases in complex with the substrate, enabling detailed structural comparison. The study revealed a monomeric IBV M pro, compared its structure to other M pro forms to guide substrate‑based inhibitor design, demonstrated that the Michael acceptor N3 inactivates IBV M pro and shows potent antiviral activity in chicken embryos, and that optimized analogues N27 and H16 potently inhibit SARS‑CoV M pro, establishing a feasible animal model for broad‑spectrum CoV inhibitor development.

Abstract

ABSTRACT Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M pro ), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) M pro and a severe acute respiratory syndrome CoV (SARS-CoV) M pro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M pro . A monomeric form of IBV M pro was identified for the first time in CoV M pro structures. A comparison of these two structures to other available M pro structures provides new insights for the design of substrate-based inhibitors targeting CoV M pro s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M pro and was found to demonstrate in vitro inactivation of IBV M pro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M pro .