Expedited Approach toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors

Abstract

The main protease (M^pro) of SARS-CoV-2 is a validated antiviral drug target. Several M^pro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including <b>GC376</b>, <b>boceprevir</b>, <b>calpain inhibitors II</b>, and <b>XII</b>, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, <b>23R</b> (<b>Jun8-76-3A</b>) that is structurally distinct from the canonical M^pro inhibitor <b>GC376</b>. Significantly, <b>23R</b> is highly selective compared with covalent inhibitors such as <b>GC376</b>, especially toward host proteases. The cocrystal structure of SARS-CoV-2 M^pro with <b>23R</b> revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered <b>23R</b>, one of the most potent and selective noncovalent SARS-CoV-2 M^pro inhibitors reported to date, and a novel binding pocket in M^pro that can be explored for inhibitor design.