An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity
Naoya Kitamura, Michael Dominic Sacco, Chunlong Ma, Yanmei Hu, Julia Alma Townsend, Xiangzhi Meng, Fushun Zhang, Xiujun Zhang, Adis Kukuljac, Michael Thomas Marty, D. Schultz, Sara R. Cherry, Yan Xiang, Yu Chen, Jun Wang · bioRxiv (Cold Spring Harbor Laboratory) · 2020
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 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 pro inhibitors. The most potent compound 23R had 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 of 23R , which inhibits SARS-CoV-2 M pro and SARS-CoV-2 viral replication with an IC 50 of 0.31 μM and EC 50 of 1.27 μM, respectively. The binding and specificity of 23R to SARS-CoV-2 M pro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 M pro with 23R revealed 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 pro inhibitors reported to date and a novel binding pocket that can be explored for M pro inhibitor design.