Structural basis of main proteases of coronavirus bound to drug candidate PF-07321332 (preprint)

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genus of coronaviruses is the substrate binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332 developed by Pfizer is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here we report three crystal structures of main protease of SARS-CoV-2, SARS-CoV and MERS-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of main protease harbors multiple inhibitor binding sites, where PF-07321332 occupies subsites S1, S2 and S4 and appears more restricted compared with other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of main proteases from different coronaviruses. Given the importance of main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals.

Crystal structure of SARS-CoV-2 main protease in complex with a Chinese herb inhibitor shikonin (preprint)

Main protease (Mpro, also known as 3CLpro) has a major role in the replication of coronavirus life cycle and is one of the most important drug targets for anticoronavirus agents. Here we report the crystal structure of main protease of SARS-CoV-2 bound to a previously identified Chinese herb inhibitor shikonin at 2.45 angstrom resolution. Although the structure revealed here shares similar overall structure with other published structures, there are several key differences which highlight potential features that could be exploited. The catalytic dyad His41-Cys145 undergoes dramatic conformational changes, and the structure reveals an unusual arrangement of oxyanion loop stabilized by the substrate. Binding to shikonin and binding of covalent inhibitors show different binding modes, suggesting a diversity in inhibitor binding. As we learn more about different binding modes and their structure-function relationships, it is probable that we can design more effective and specific drugs with high potency that can serve as effect SARS-CoV-2 anti-viral agents.

Structure of SARS-CoV-2 main protease in the apo state reveals the inactive conformation (preprint)

Mpro is of considerable interest as a drug target in the treatment of COVID-19 since the proteolytic activity of this viral protease is essential for viral replication. Here we report the first insight of the structure Mpro for SARS-CoV-2 in the inactive conformation under conditions close to the physiological state (pH 7.5) to an overall resolution of 1.9 [A]. The comparisons of Mpro in different states reveal that substrate binding site and the active site are more flexible in the inactive conformation than that in the active conformations. Notably, compared with the active conformation of the apo state structure in pH7.6 of SARS, the SARS-CoV-2 apo state is in the inactive conformation under condition close to physiological state (pH7.5). Two water molecules are present in the oxyanion hole in our apo state structure, whereas in the ligand-bound structure, water molecular is absence in the same region. This structure provides novel and important insights that have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational, structure-based, approaches for the design of specific SARS-CoV-2 ligands as new therapeutic agents.