The SARS-CoV-2 main protease (M ^pro ) is essential for viral replication and is a primary target for COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, target the M ^pro active site to block viral polyprotein cleavage and thus replication. However, drug resistance mutations at the active site residue Glu166 (E166) have emerged during in vitro selection studies, raising concerns about the durability of current antiviral strategies. Here, we investigate the molecular basis of drug resistance conferred by E166A and E166V mutations against nirmatrelvir and the related PF-00835231, individually and in combination with the distal mutation L50F. We found that E166 mutations reduce nirmatrelvir potency by up to 3,000-fold while preserving substrate cleavage, with catalytic efficiency reduced by only up to twofold. This loss of catalytic efficiency was compensated for by the addition of L50F in the double-mutant variants. We have determined three cocrystal structures of the E166 variants (E166A, E166V, and E166V/L50F) bound to PF-00835231. Comparison of these structures with wild-type enzyme demonstrated that E166 is crucial for dimerization and for shaping the substrate-binding S1 pocket. Our findings highlight the mutability of E166, a prime site for resistance for inhibitors that leverage direct interactions with this position, and the potential emergence of highly resistant and active variants in combination with the compensatory mutation L50F. These insights support the design of inhibitors that target conserved protease features and avoid E166 side-chain interactions to minimize susceptibility to resistance. IMPORTANCE: Drug resistance remains a great challenge to modern medicine. This study investigates SARS-CoV-2 main protease variants E166A and E166V which confer nirmatrelvir resistance. These variants can retain considerable enzymatic activity through combination with the compensatory mutation L50F. For single- and double-mutant variant enzymes, we assessed catalytic efficiency, measured loss in potency for nirmatrelvir and its analog PF-00835231, and cocrystallized with inhibitors to investigate drug resistance caused by these mutations. Our results contribute toward understanding of molecular mechanisms of resistance and combinations of mutations, which pushes toward resistance-thwarting inhibitor design. These principles also apply broadly to many quickly evolving drug targets in infectious diseases.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (M^pro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 M^pro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 M^pro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.

Enterovirus-D68 (EV68) has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Unfortunately, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. For other viral proteases, we have demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. However, the structural characterization of EV68-3C protease bound to its substrates has been lacking. Here, we have determined the substrate specificity of EV68-3C protease through molecular modeling, molecular dynamics (MD) simulations, and co-crystal structures. Molecular models enabled us to successfully characterize the conserved hydrogen-bond networks between EV68-3C protease and the peptides corresponding to the viral cleavage sites. In addition, co-crystal structures we determined have revealed substrate-induced conformational changes of the protease which involved new interactions, primarily surrounding the S1 pocket. We calculated the substrate envelope, the three-dimensional consensus volume occupied by the substrates within the active site. With the elucidation of the EV68-3C protease substrate envelope, we evaluated how 3C protease inhibitors, AG7088 and SG-85, fit within the active site to predict potential resistance mutations.

The coronaviral main protease (M ^pro ) has been the subject of various biochemical and structural studies and a drug target against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. SARS-CoV-2 M ^pro is active as a dimer, but despite apparent cooperativity in catalytic activity, how the two distal active sites communicate and modulate binding and/or catalysis is unclear. Here, we have investigated the interplay between cooperativity, dimerization, and substrate cleavage in SARS-CoV-2 M ^pro through a combination of enzymatic assays, crystal structures, and protein characterization. To disentangle the contribution of each active site to the observed enzymatic activity, we developed a cleavage assay involving heterodimers of active and inactive (catalytic residue mutated or inhibitor-bound) monomers. Notably, we found that heterodimerization increased cleavage efficiency per active monomer. In addition, we mapped a network of critical residues bridging the two active sites and probed this network through engineered mutations. By dissecting the cooperativity and communication between the active sites, we provide insights into the M ^pro reaction cycle and functional significance of its dimeric architecture.

Enterovirus-D68 (EV68) continues to present as a global health issue causing respiratory illness and outbreaks associated with long-lasting neurological disease, with no antivirals or specific treatment options. The development of antiviral therapeutics, such as small-molecule inhibitors that target conserved proteins like the enteroviral 3C protease, remains to be achieved. While various 3C inhibitors have been investigated, their design does not consider the potential emergence of drug resistance mutations. For other antivirals where resistance has been a challenge, we have demonstrated that the likelihood of resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of the target. Here, we characterize a series of 3C inhibitors against EV68-3C protease through enzyme inhibition, protein crystallography, and structural analysis. We have determined and analyzed three high-resolution inhibitor-bound crystal structures of EV68-3C protease, which revealed possible sites of resistance mutations, a key structural water molecule conserved during ligand binding, and the conformational flexibility of the catalytic histidine H40. This structural analysis combined with enzymatic assays provides insights for the rational design of inhibitors that are robust against resistance toward developing antiviral treatments for EV68 infections.

Coronaviruses include various strains that reside in natural animal reservoirs, with zoonotic transmission posing risks to both domesticated animals and human health. Recent efforts to address coronavirus infections have focused on developing inhibitors targeting the main protease (M^pro), some of which exhibit potential broad-spectrum efficacy. This study presents crystal structures of four clinically relevant inhibitors—GC376, PF-00835231, nirmatrelvir, and ibuzatrelvir—bound to M^pro from the feline coronavirus strain FECV-UU23. Structural analysis identified distinct FECV-specific features within the active site where these inhibitors bind and revealed S4 loop as a susceptible structural region essential for the enhanced binding of inhibitors in UU23 M^pro. We therefore propose to incorporate sterically constrained, functionally tailored heterocyclic moieties at the P3 site of known inhibitors which can optimally engage Q187, P188, and S189 residues of the S4 loop. The findings presented enhance understanding of inhibitor specificity and reinforce the promise of these inhibitor scaffolds for developing antivirals against feline coronavirus strains, with possible applications in broad-spectrum coronavirus therapy.