New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (M^pro or 3CL^pro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 M^pro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against M^pros from multiple coronaviruses. Crystal structures of M^pros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of M^pros from different coronaviruses. In view of the importance of M^pro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.

The ongoing spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused hundreds of millions of cases and millions of victims worldwide with serious consequences to global health and economies. Although many vaccines protecting against SARS-CoV-2 are currently available, constantly emerging new variants necessitate the development of alternative strategies for prevention and treatment of COVID-19. Inhibitors that target the main protease (M^pro) of SARS-CoV-2, an essential enzyme that promotes viral maturation, represent a key class of antivirals. Here, we showed that a peptidomimetic compound with benzothiazolyl ketone as warhead, YH-53, is an effective inhibitor of SARS-CoV-2, SARS-CoV, and MERS-CoV M^pros. Crystal structures of M^pros from SARS-CoV-2, SARS-CoV, and MERS-CoV bound to the inhibitor YH-53 revealed a unique ligand-binding site, which provides new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures defined the key molecular determinants required for inhibition and illustrate the binding mode of M^pros from other coronaviruses. In consideration of the important role of M^pro in developing antivirals against coronaviruses, insights derived from this study should add to the design of pan-coronaviral M^pro inhibitors that are safer and more effective.

Preventing the spread of SARS-CoV-2 and its variants is crucial in the fight against COVID-19. Inhibition of the main protease (M^pro) of SARS-CoV-2 is the key to disrupting viral replication, making M^pro a promising target for therapy. PF-07321332 and shikonin have been identified as effective broad-spectrum inhibitors of SARS-CoV-2 M^pro. The crystal structures of SARS-CoV-2 M^pro bound to PF-07321332 and shikonin have been resolved in previous studies. However, the exact mechanism regarding how SARS-CoV-2 M^pro mutants impact their binding modes largely remains to be investigated. In this study, we expressed a SARS-CoV-2 M^pro mutant, carrying the D48N substitution, representing a class of mutations located near the active sites of M^pro. The crystal structures of M^pro D48N in complex with PF-07321332 and shikonin were solved. A detailed analysis of the interactions between M^pro D48N and two inhibitors provides key insights into the binding pattern and its structural determinants. Further, the binding patterns of the two inhibitors to M^pro D48N mutant and wild-type M^pro were compared in detail. This study illustrates the possible conformational changes when the M^pro D48N mutant is bound to inhibitors. Structural insights derived from this study will inform the development of new drugs against novel coronaviruses.

M ^pro 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 M ^pro 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 Å. The comparisons of M ^pro 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.

Main protease (M ^pro , 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.