A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiological agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, we report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to our knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.

The genus Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (M^pros), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of M^pro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV M^pros, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.

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 .