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.

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.