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Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design

Wioletta Rut, Zongyang Lv, Mikolaj Zmudzinski, Stephanie Patchett, Digant Nayak, Scott J. Snipas, Farid El Oualid, Tony T. Huang, Miklos Bekes, Marcin Drag, Shaun K. Olsen
doi: https://doi.org/10.1101/2020.04.29.068890
Wioletta Rut
1Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
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  • For correspondence: wioletta.rut@pwr.edu.pl miklosbekes@icloud.com olsens@uthscsa.edu marcin.drag@pwr.edu.pl
Zongyang Lv
2Department of Biochemistry & Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA
7Department of Biochemistry & Structural Biology University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229 USA
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Mikolaj Zmudzinski
1Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
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Stephanie Patchett
3Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
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Digant Nayak
2Department of Biochemistry & Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA
7Department of Biochemistry & Structural Biology University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229 USA
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Scott J. Snipas
4Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
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Farid El Oualid
5UbiQ Bio B.V., 1098 XH, Amsterdam, The Netherlands
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Tony T. Huang
3Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
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Miklos Bekes
6Independent Consultant
8Arvinas, Inc., 5 Science Park, New Haven, CT, 06511, USA
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  • For correspondence: wioletta.rut@pwr.edu.pl miklosbekes@icloud.com olsens@uthscsa.edu marcin.drag@pwr.edu.pl
Marcin Drag
1Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
4Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
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  • For correspondence: wioletta.rut@pwr.edu.pl miklosbekes@icloud.com olsens@uthscsa.edu marcin.drag@pwr.edu.pl
Shaun K. Olsen
2Department of Biochemistry & Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA
7Department of Biochemistry & Structural Biology University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229 USA
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  • For correspondence: wioletta.rut@pwr.edu.pl miklosbekes@icloud.com olsens@uthscsa.edu marcin.drag@pwr.edu.pl
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Abstract

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.

Competing Interest Statement

F.E.O. declares competing financial interests as co-founders and shareholder of UbiQ Bio BV. M.B. is an employee and shareholder of Arvinas, Inc. The remaining authors declare no competing interests.

Footnotes

  • This version is updated with crystal structures of inhibitors with SARS-CoV-2-PLpro and biochemistry data.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 15, 2020.
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Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design
Wioletta Rut, Zongyang Lv, Mikolaj Zmudzinski, Stephanie Patchett, Digant Nayak, Scott J. Snipas, Farid El Oualid, Tony T. Huang, Miklos Bekes, Marcin Drag, Shaun K. Olsen
bioRxiv 2020.04.29.068890; doi: https://doi.org/10.1101/2020.04.29.068890
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Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design
Wioletta Rut, Zongyang Lv, Mikolaj Zmudzinski, Stephanie Patchett, Digant Nayak, Scott J. Snipas, Farid El Oualid, Tony T. Huang, Miklos Bekes, Marcin Drag, Shaun K. Olsen
bioRxiv 2020.04.29.068890; doi: https://doi.org/10.1101/2020.04.29.068890

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