Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Brian C. Sanders; Suman Pokhrel; Audrey D. Labbe; Irimpan I. Mathews; Connor J. Cooper; Russell B. Davidson; Gwyndalyn Phillips; Kevin L. Weiss; Qiu Zhang; Hugh O’Neill; Manat Kaur; Jurgen G. Schmidt; Walter Reichard; Surekha Surendranathan; Jyothi Parvathareddy; Lexi Phillips; Christopher Rainville; David E. Sterner; Desigan Kumaran; Babak Andi; Gyorgy Babnigg; Nigel W. Moriarty; Paul D. Adams; Andrzej Joachimiak; Brett L. Hurst; Suresh Kumar; Tauseef R. Butt; Colleen B. Jonsson; Lori Ferrins; Soichi Wakatsuki; Stephanie Galanie; Martha S. Head; Jerry M. Parks

doi:10.1038/s41467-023-37254-w

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Brian C. Sanders, Suman Pokhrel, Audrey D. Labbe, Irimpan I. Mathews, Connor J. Cooper, Russell B. Davidson, Gwyndalyn Phillips, Kevin L. Weiss, Qiu Zhang, Hugh O’Neill, Manat Kaur, Jurgen G. Schmidt, Walter Reichard, Surekha Surendranathan, Jyothi Parvathareddy, Lexi Phillips, Christopher Rainville, David E. Sterner, Desigan Kumaran, Babak AndiGyorgy Babnigg, Nigel W. Moriarty, Paul D. Adams, Andrzej Joachimiak, Brett L. Hurst, Suresh Kumar, Tauseef R. Butt, Colleen B. Jonsson, Lori Ferrins, Soichi Wakatsuki, Stephanie Galanie, Martha S. Head, Jerry M. Parks

Research output: Contribution to journal › Article › peer-review

34 Scopus citations

Abstract

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with k_inact/K_I = 9,600 M⁻¹ s⁻¹, achieves sub-μM EC₅₀ values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Original language	English
Article number	1733
Journal	Nature Communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-37254-w
State	Published - Dec 2023

Funding

We thank Paul Abraham and Richard Giannone for assistance with mass spectrometry and Yunqiao (Joseph) Pu for assistance with nuclear magnetic resonance spectroscopy. We also thank Daniel Fernandez and the Macromolecular Structure Knowledge Center at Stanford for providing equipment for crystallography. We thank Bernd Meibohm for helpful discussions. This work was supported by the U.S. Department of Energy (DOE) Office of Science through the National Virtual Biotechnology Laboratory, a consortium of DOE national laboratories focused on response to COVID-19, with funding provided by the Coronavirus CARES Act. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Funding for this project was provided in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract HHSN272201700060C and 75N93022C00035 (A.J.). A.L. was supported in part by the Laboratory Directed Research and Development Program at Oak Ridge National Laboratory (ORNL) and through an appointment to the Science Education and Workforce Development Programs at ORNL, administered by Oak Ridge Institute for Science and Education (ORISE) through the U.S. DOE. Protein production was supported by the ORNL Center for Structural Molecular Biology funded by the DOE OBER. B.C.S. and J.M.P. also received funding from the Technology Innovation Program at ORNL. This research used resources at the Spallation Neutron Source, a U.S. Department of Energy Office of Science User Facility operated by ORNL, and at the Compute and Data Environment for Science (CADES) at ORNL, which is managed by UT Battelle, LLC, for DOE under contract DE-AC05–00OR22725. We thank Paul Abraham and Richard Giannone for assistance with mass spectrometry and Yunqiao (Joseph) Pu for assistance with nuclear magnetic resonance spectroscopy. We also thank Daniel Fernandez and the Macromolecular Structure Knowledge Center at Stanford for providing equipment for crystallography. We thank Bernd Meibohm for helpful discussions. This work was supported by the U.S. Department of Energy (DOE) Office of Science through the National Virtual Biotechnology Laboratory, a consortium of DOE national laboratories focused on response to COVID-19, with funding provided by the Coronavirus CARES Act. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Funding for this project was provided in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract HHSN272201700060C and 75N93022C00035 (A.J.). A.L. was supported in part by the Laboratory Directed Research and Development Program at Oak Ridge National Laboratory (ORNL) and through an appointment to the Science Education and Workforce Development Programs at ORNL, administered by Oak Ridge Institute for Science and Education (ORISE) through the U.S. DOE. Protein production was supported by the ORNL Center for Structural Molecular Biology funded by the DOE OBER. B.C.S. and J.M.P. also received funding from the Technology Innovation Program at ORNL. This research used resources at the Spallation Neutron Source, a U.S. Department of Energy Office of Science User Facility operated by ORNL, and at the Compute and Data Environment for Science (CADES) at ORNL, which is managed by UT Battelle, LLC, for DOE under contract DE-AC05–00OR22725.

Funders	Funder number
CADES	DE-AC05–00OR22725
Data Environment for Science
Macromolecular Structure Knowledge Center
National Virtual Biotechnology Laboratory
National Institutes of Health
U.S. Department of Energy
U.S. Department of Health and Human Services	75N93022C00035, HHSN272201700060C
National Institute of General Medical Sciences	P30GM133894
National Institute of Allergy and Infectious Diseases
Office of Science
Basic Energy Sciences	DE-AC02-76SF00515
Biological and Environmental Research
Oak Ridge National Laboratory
Oak Ridge Institute for Science and Education

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Sanders, B. C., Pokhrel, S., Labbe, A. D., Mathews, I. I., Cooper, C. J., Davidson, R. B., Phillips, G., Weiss, K. L., Zhang, Q., O’Neill, H., Kaur, M., Schmidt, J. G., Reichard, W., Surendranathan, S., Parvathareddy, J., Phillips, L., Rainville, C., Sterner, D. E., Kumaran, D., ... Parks, J. M. (2023). Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2. Nature Communications, 14(1), Article 1733. https://doi.org/10.1038/s41467-023-37254-w

@article{c72ccbba6e634800a83072c9379eced0,

title = "Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2",

abstract = "Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M−1 s−1, achieves sub-μM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.",

author = "Sanders, {Brian C.} and Suman Pokhrel and Labbe, {Audrey D.} and Mathews, {Irimpan I.} and Cooper, {Connor J.} and Davidson, {Russell B.} and Gwyndalyn Phillips and Weiss, {Kevin L.} and Qiu Zhang and Hugh O{\textquoteright}Neill and Manat Kaur and Schmidt, {Jurgen G.} and Walter Reichard and Surekha Surendranathan and Jyothi Parvathareddy and Lexi Phillips and Christopher Rainville and Sterner, {David E.} and Desigan Kumaran and Babak Andi and Gyorgy Babnigg and Moriarty, {Nigel W.} and Adams, {Paul D.} and Andrzej Joachimiak and Hurst, {Brett L.} and Suresh Kumar and Butt, {Tauseef R.} and Jonsson, {Colleen B.} and Lori Ferrins and Soichi Wakatsuki and Stephanie Galanie and Head, {Martha S.} and Parks, {Jerry M.}",

note = "Publisher Copyright: {\textcopyright} 2023, UT-Battelle, LLC.",

year = "2023",

month = dec,

doi = "10.1038/s41467-023-37254-w",

language = "English",

volume = "14",

journal = "Nature Communications",

issn = "2041-1723",

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Sanders, BC, Pokhrel, S, Labbe, AD, Mathews, II, Cooper, CJ, Davidson, RB, Phillips, G, Weiss, KL , Zhang, Q, O’Neill, H, Kaur, M, Schmidt, JG, Reichard, W, Surendranathan, S, Parvathareddy, J, Phillips, L, Rainville, C, Sterner, DE, Kumaran, D, Andi, B, Babnigg, G, Moriarty, NW, Adams, PD, Joachimiak, A, Hurst, BL, Kumar, S, Butt, TR, Jonsson, CB, Ferrins, L, Wakatsuki, S, Galanie, S, Head, MS & Parks, JM 2023, 'Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2', Nature Communications, vol. 14, no. 1, 1733. https://doi.org/10.1038/s41467-023-37254-w

TY - JOUR

T1 - Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

AU - Sanders, Brian C.

AU - Pokhrel, Suman

AU - Labbe, Audrey D.

AU - Mathews, Irimpan I.

AU - Cooper, Connor J.

AU - Davidson, Russell B.

AU - Phillips, Gwyndalyn

AU - Weiss, Kevin L.

AU - Zhang, Qiu

AU - O’Neill, Hugh

AU - Kaur, Manat

AU - Schmidt, Jurgen G.

AU - Reichard, Walter

AU - Surendranathan, Surekha

AU - Parvathareddy, Jyothi

AU - Phillips, Lexi

AU - Rainville, Christopher

AU - Sterner, David E.

AU - Kumaran, Desigan

AU - Andi, Babak

AU - Babnigg, Gyorgy

AU - Moriarty, Nigel W.

AU - Adams, Paul D.

AU - Joachimiak, Andrzej

AU - Hurst, Brett L.

AU - Kumar, Suresh

AU - Butt, Tauseef R.

AU - Jonsson, Colleen B.

AU - Ferrins, Lori

AU - Wakatsuki, Soichi

AU - Galanie, Stephanie

AU - Head, Martha S.

AU - Parks, Jerry M.

PY - 2023/12

Y1 - 2023/12

N2 - Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M−1 s−1, achieves sub-μM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

AB - Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M−1 s−1, achieves sub-μM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

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U2 - 10.1038/s41467-023-37254-w

DO - 10.1038/s41467-023-37254-w

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JO - Nature Communications

JF - Nature Communications

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M1 - 1733

ER -

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

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