The S1’‐S3’ Pocket of the SARS‐CoV‐2 Main Protease Is Critical for Substrate Selectivity and Can Be Targeted with Covalent Inhibitors

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a well‐characterized target for antiviral drug discovery. To date, most antiviral drug discovery efforts have focused on the S4‐S1’ pocket of Mpro; however, it is still unclear whether the S1’‐S3’ pocket per se can serve as a new site for drug discovery. In this study, the S1’‐S3’ pocket of Mpro was found to differentially recognize viral peptidyl substrates; for instance, S3’ in Mpro strongly favors Phe or Trp, and S1’ favors Ala. The peptidyl inhibitor D‐4‐77, which possesses an α‐bromoacetamide warhead, was discovered to be a promising inhibitor of Mpro, with an IC50 of 0.95 µM and an antiviral EC50 of 0.49 µM. The Mpro/inhibitor co‐crystal structure confirmed the binding mode of the inhibitor to the S1’‐S3’ pocket and revealed a covalent mechanism. In addition, D‐4‐77 functions as an immune protectant and suppresses SARS‐CoV‐2 Mpro‐induced antagonism of the host NF‐κB innate immune response. These findings indicate that the S1’‐S3’ pocket of SARS‐CoV‐2 Mpro is druggable, and moreover, inhibiting SARS‐CoV‐2 Mpro can simultaneously protect human innate immunity and inhibit virion assembly.