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.

During the continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November of that year. Along with its sublineages, it has maintained a prominent role ever since. The Nsp5 main protease (M^pro) of the Omicron virus is characterized by a single dominant mutation, P132H. Here we determined the X-ray crystal structures of the P132H mutant (or O-M^pro) as a free enzyme and in complex with the M^pro inhibitor, the alpha-ketoamide 13b-K, and we conducted enzymological, biophysical, as well as theoretical studies to characterize the O-M^pro. We found that O-M^pro has a similar overall structure and binding with 13b-K; however, it displays lower enzymatic activity and lower thermal stability compared to the WT-M^pro (with “WT” referring to the prototype strain). Intriguingly, the imidazole ring of His132 and the carboxylate plane of Glu240 are in a stacked configuration in the X-ray structures determined here. Empirical folding free energy calculations suggest that the O-M^pro dimer is destabilized relative to the WT-M^pro due to less favorable van der Waals interactions and backbone conformations in the individual protomers. All-atom continuous constant-pH molecular dynamics (MD) simulations reveal that His132 and Glu240 display coupled titration. At pH 7, His132 is predominantly neutral and in a stacked configuration with respect to Glu240 which is charged. In order to examine whether the Omicron mutation eases the emergence of further M^pro mutations, we also analyzed the P132H+T169S double mutant, which is characteristic of the BA.1.1.2 lineage. However, we found little evidence of a correlation between the two mutation sites.

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).

The SARS-CoV-2 main protease (M^pro) is a well-established target for antiviral drug development. One such inhibitor, nirmatrelvir, in combination with ritonavir as a booster, has already been introduced into the market, under the name Paxlovid. However, being an RNA virus, SARS-CoV-2 is prone to the emergence of resistance mutations. A number of such mutations have been characterized, although they have not yet been shown to play a significant role in clinical settings; these include S144A, E166V, H172Y, and Q189K. We recombinantly produced these mutants and studied the corresponding proteins using X-ray crystallography, enzymology, and biophysical approaches. We discuss the potential of each mutant to lead to a widespread nirmatrelvir resistance scenario. We also demonstrate that one of our own inhibitors (13b-K), while showing some degree of cross-resistance with nirmatrelvir, exhibits much higher inhibitory activity against the M^pro carrying the E166V mutation.

Viral proteases represent validated targets for direct-acting antivirals and the treatment of associated infections. In co-crystal structures of M ^pro of SARS-CoV-2 with peptidomimetic inhibitors, we noticed a spatial proximity of sidechains filling the S1’ and S2 pockets, as well as those filling S3 and S1 pockets. To enhance molecular rigidity, the proximal residues were conformationally fixed by macrocyclization. We report the synthesis of two macrocyclic series, i.e. exocyclic nitriles with linked P3 and P1 residues and endocyclic α-ketoamides with linked P1’ and P2 residues, and characterize their binding modes and bioactivities. The 17-membered macrocyclic α-ketoamide 20 f inhibited M ^pro (IC₅₀ = 370 nM) and exerted anti-SARS-CoV-2 effects (EC₅₀ = 1.9 μM). Leveraging structural similarities between M ^pro and the 3C ^pro of enterovirus D68, we describe with two co-crystal structures how α-ketoamide macrocycles bound to and inhibited the enteroviral protease. Notably, 20 f exhibited very potent antiviral activities with EC₅₀‘s of 33, 133, and 146 nM against EV-D68, EV-A71, and CVB3, respectively. The study demonstrates how broad-spectrum activity can be achieved with direct-acting antivirals.