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

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 .