Structures of two coronavirus main proteases: Implications for substrate binding and antiviral drug design

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. 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.