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The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir

View ORCID ProfileDirk Jochmans, Cheng Liu, Kim Donckers, Antitsa Stoycheva, View ORCID ProfileSandro Boland, Sarah K Stevens, Chloe De Vita, View ORCID ProfileBert Vanmechelen, View ORCID ProfilePiet Maes, View ORCID ProfileBettina Trüeb, Nadine Ebert, View ORCID ProfileVolker Thiel, View ORCID ProfileSteven De Jonghe, View ORCID ProfileLaura Vangeel, Dorothée Bardiot, View ORCID ProfileAndreas Jekle, View ORCID ProfileLawrence M Blatt, Leonid Beigelman, Julian A Symons, Pierre Raboisson, Patrick Chaltin, View ORCID ProfileArnaud Marchand, View ORCID ProfileJohan Neyts, Jerome Deval, View ORCID ProfileKoen Vandyck
doi: https://doi.org/10.1101/2022.06.07.495116
Dirk Jochmans
1KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Herestraat 49, 3000 Leuven, Belgium
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  • For correspondence: dirk.jochmans@kuleuven.be
Cheng Liu
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Kim Donckers
1KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Herestraat 49, 3000 Leuven, Belgium
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Antitsa Stoycheva
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Sandro Boland
3CISTIM Leuven vzw, Gaston Geenslaan 2, 3001 Leuven, Belgium
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Sarah K Stevens
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Chloe De Vita
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Bert Vanmechelen
4KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Clinical & Epidemiological Virology, Herestraat 49, Leuven, 3000, Belgium
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Piet Maes
4KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Clinical & Epidemiological Virology, Herestraat 49, Leuven, 3000, Belgium
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Bettina Trüeb
5Institute of Virology and Immunology, University of Bern, 3012, Bern, Switzerland; Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
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Nadine Ebert
5Institute of Virology and Immunology, University of Bern, 3012, Bern, Switzerland; Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
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Volker Thiel
5Institute of Virology and Immunology, University of Bern, 3012, Bern, Switzerland; Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
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Steven De Jonghe
1KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Herestraat 49, 3000 Leuven, Belgium
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Laura Vangeel
1KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Herestraat 49, 3000 Leuven, Belgium
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Dorothée Bardiot
3CISTIM Leuven vzw, Gaston Geenslaan 2, 3001 Leuven, Belgium
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Andreas Jekle
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Lawrence M Blatt
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Leonid Beigelman
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Julian A Symons
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Pierre Raboisson
6Aligos Belgium BV, Gaston Geenslaan 1, 3001 Leuven, Belgium
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Patrick Chaltin
7Centre for Drug Design and Discovery (CD3), KU Leuven, Gaston Geenslaan 2, 3001 Leuven, Belgium; CISTIM Leuven vzw, Gaston Geenslaan 2, 3001 Leuven, Belgium
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Arnaud Marchand
3CISTIM Leuven vzw, Gaston Geenslaan 2, 3001 Leuven, Belgium
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Johan Neyts
8KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Herestraat 49, 3000 Leuven, Belgium and Global Virus Network (GVN)
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Jerome Deval
2Aligos Therapeutics, Inc., 1 Corporate Dr., 2nd Floor, South San Francisco, CA, USA
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Koen Vandyck
6Aligos Belgium BV, Gaston Geenslaan 1, 3001 Leuven, Belgium
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Abstract

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6- to 72-fold). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting.

Competing Interest Statement

The authors declare the following financial interests / personal relationships which may be considered as potential competing interests: - Koen Vandyck and Pierre Raboisson are employees of Aligos Belgium BV - Cheng Liu, Antitsa Stoycheva, Sarah K Stevens, Chloe De Vita, Andreas Jekle, Lawrence M Blatt, Leonid Beigelman, Julian A Symons and Jerome Deval are employees of Aligos Therapeutics, Inc. - A patent application on ALG-097161 is pending

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 07, 2022.
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The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir
Dirk Jochmans, Cheng Liu, Kim Donckers, Antitsa Stoycheva, Sandro Boland, Sarah K Stevens, Chloe De Vita, Bert Vanmechelen, Piet Maes, Bettina Trüeb, Nadine Ebert, Volker Thiel, Steven De Jonghe, Laura Vangeel, Dorothée Bardiot, Andreas Jekle, Lawrence M Blatt, Leonid Beigelman, Julian A Symons, Pierre Raboisson, Patrick Chaltin, Arnaud Marchand, Johan Neyts, Jerome Deval, Koen Vandyck
bioRxiv 2022.06.07.495116; doi: https://doi.org/10.1101/2022.06.07.495116
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The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir
Dirk Jochmans, Cheng Liu, Kim Donckers, Antitsa Stoycheva, Sandro Boland, Sarah K Stevens, Chloe De Vita, Bert Vanmechelen, Piet Maes, Bettina Trüeb, Nadine Ebert, Volker Thiel, Steven De Jonghe, Laura Vangeel, Dorothée Bardiot, Andreas Jekle, Lawrence M Blatt, Leonid Beigelman, Julian A Symons, Pierre Raboisson, Patrick Chaltin, Arnaud Marchand, Johan Neyts, Jerome Deval, Koen Vandyck
bioRxiv 2022.06.07.495116; doi: https://doi.org/10.1101/2022.06.07.495116

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