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

Kitamura, N.; Sacco, M. D.; Ma, C.; Hu, Y.; Townsend, J.; Meng, X.; Zhang, F.; Zhang, X.; Kukuljac, A.; Marty, M.; Schultz, D.; Cherry, S.; Xiang, Y.; Chen, Y.; Wang, J.

doi:10.1101/2020.12.19.423537

bioRxiv · 10.1101/2020.12.19.423537

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

Kitamura, N.·Sacco, M. D.·Ma, C.·Hu, Y.·Townsend, J.·Meng, X.·Zhang, F.·Zhang, X.·Kukuljac, A.·Marty, M.·Schultz, D.·Cherry, S.·Xiang, Y.·Chen, Y.·Wang, J.

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Abstract

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro 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 Mpro 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 Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + 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 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 M and EC50 of 1.27 M, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -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 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

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Kitamura, N., Sacco, M. D., Ma, C., Hu, Y., Townsend, J., Meng, X., Zhang, F., Zhang, X., Kukuljac, A., Marty, M., Schultz, D., Cherry, S., Xiang, Y., Chen, Y., Wang, J.. 2020-12-20. An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity. https://doi.org/10.1101/2020.12.19.423537

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