Crystal structures of coronaviral main proteases in complex with the non-covalent inhibitor X77

Main proteases (M^pros) are a class of conserved cysteine hydrolases among coronaviruses and play a crucial role in viral replication. Therefore, M^pros are ideal targets for the development of pan-coronavirus drugs. X77, previously developed against SARS-CoV M^pro, was repurposed as a non-covalent tight binder inhibitor against SARS-CoV-2 M^pro during COVID-19 pandemic. Many novel inhibitors with favorable efficacy have been discovered using X77 as a reference, suggesting that X77 could be a valuable scaffold for drug design. However, the broad-spectrum performance of X77 and underlying mechanism remain less understood. Here, we reported the crystal structures of M^pros from SARS-CoV-2, SARS-CoV, and MERS-CoV, and several M^pro mutants from SARS-CoV-2 variants bound to X77. A detailed analysis of these structures revealed key structural determinants essential for interaction and elucidated the binding modes of X77 with different coronaviral M^pros. The potencies of X77 against these investigated M^pros were further evaluated through molecular dynamic simulation and binding free energy calculation. These data provide molecular insights into broad-spectrum inhibition against coronaviral M^pros by X77 and the similarities and differences of X77 when bound to various M^pros, which will promote X77-based design of novel antivirals with broad-spectrum efficacy against different coronaviruses and SARS-CoV-2 variants.