The main protease (M^pro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the M^pro. Starting from crystal structures of the M^pro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the M^pro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC₅₀ of 13 nM versus the recombinant M^pro, and similar potency was observed for its P1′ N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 M^pro inhibition, we also explored the activity of MG-78 against the M^pro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 M^pro), moderate (1.45 µM, Coxsackievirus 3C^pro), and relatively poor (6.7 µM, enterovirus A71 3C^pro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus M^pros but further optimization would be needed to target enterovirus 3C^pros efficiently.

The SARS-CoV-2 main protease (M^pro), an enzyme essential for viral replication and lacking a human homologue, has emerged as a highly attractive target for the development of novel antiviral agents. Although several M^pro inhibitors have been developed - some receiving regulatory approval - their use is sometimes limited by drug-drug interactions. In this study, we designed and synthesized peptidomimetic SARS-CoV-2 M^pro inhibitors incorporating a novel thiazolyl 4-carboxylate ketone warhead, previously employed by our group in the development of cytosolic phospholipase A₂ inhibitors. The synthesized compounds were evaluated for their in vitro inhibitory potency against SARS-CoV-2 M^pro, and a highly potent M^pro inhibitor (GK730) was identified (IC₅₀ 5.75 nM). The melting temperature of the M^pro-GK730 complex revealed high stability, consistent with the high inhibitory potency. The X-ray crystal structures of inhibitors GK729 and GK730 bound to M^pro were determined, providing insights into the binding interactions and mechanism of action. Studies on the host cell proteases cathepsin B and L showed that GK730 did not inhibit cathepsin B, while exhibited weak inhibition of cathepsin L. Furthermore, GK730 demonstrated an EC₅₀ value of 5.70 μM against a wild-type SARS-CoV-2 strain in Vero E6 cells and minimal cytotoxicity (CC₅₀ value greater than 100 μM).