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

Abstract

Main proteases (Mpros) are a class of conserved cysteine hydrolases among coronaviruses and play a crucial role in viral replication. Therefore, Mpros are ideal targets for the development of pan-coronavirus drugs. X77, previously developed against SARS-CoV Mpro, was repurposed as a non-covalent tight binder inhibitor against SARS-CoV-2 Mpro 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 Mpros from SARS-CoV-2, SARS-CoV, and MERS-CoV, and several Mpro 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 Mpros. The potencies of X77 against these investigated Mpros were further evaluated through molecular dynamic simulation and binding free energy calculation. These data provide molecular insights into broad-spectrum inhibition against coronaviral Mpros by X77 and the similarities and differences of X77 when bound to various Mpros, which will promote X77-based design of novel antivirals with broad-spectrum efficacy against different coronaviruses and SARS-CoV-2 variants.