An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

The main protease (M^pro) of SARS-CoV-2 is a validated antiviral drug target. Several M^proinhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent M^proinhibitors. The most potent compound23Rhad cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV M^pro+ ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 M^pro+ calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 M^pro. Lead optimization led to the discovery of23R, which inhibits SARS-CoV-2 M^proand SARS-CoV-2 viral replication with an IC₅₀of 0.31 μM and EC₅₀of 1.27 μM, respectively. The binding and specificity of23Rto SARS-CoV-2 M^prowere confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 M^prowith23Rrevealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the α-methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 M^proinhibitors reported to date and a novel binding pocket that can be explored for M^proinhibitor design.