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High-Throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

  • Morgan Dasovich
    Morgan Dasovich
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
    Department of Chemistry, Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United States
  • Junlin Zhuo
    Junlin Zhuo
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
    More by Junlin Zhuo
  • Jack A. Goodman
    Jack A. Goodman
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Ajit Thomas
    Ajit Thomas
    Johns Hopkins Drug Discovery, Baltimore, Maryland 21205, United States
    Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
    More by Ajit Thomas
  • Robert Lyle McPherson
    Robert Lyle McPherson
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Aravinth Kumar Jayabalan
    Aravinth Kumar Jayabalan
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Veronica F. Busa
    Veronica F. Busa
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
    McKusick-Nathans Department of Genetics Medicine  and  School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Shang-Jung Cheng
    Shang-Jung Cheng
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Brennan A. Murphy
    Brennan A. Murphy
    Johns Hopkins Drug Discovery, Baltimore, Maryland 21205, United States
  • Karli R. Redinger
    Karli R. Redinger
    Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio 44106, United States
  • Yousef M. O. Alhammad
    Yousef M. O. Alhammad
    Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States
  • Anthony R. Fehr
    Anthony R. Fehr
    Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States
  • Takashi Tsukamoto
    Takashi Tsukamoto
    Johns Hopkins Drug Discovery, Baltimore, Maryland 21205, United States
    Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Barbara S. Slusher
    Barbara S. Slusher
    Johns Hopkins Drug Discovery, Baltimore, Maryland 21205, United States
    Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
  • Jürgen Bosch*
    Jürgen Bosch
    Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio 44106, United States
    InterRayBio, LLC, Cleveland, Ohio 44106, United States
    *Email: [email protected]
  • Huijun Wei*
    Huijun Wei
    Johns Hopkins Drug Discovery, Baltimore, Maryland 21205, United States
    Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
    *Email: [email protected]
    More by Huijun Wei
  • , and 
  • Anthony K. L. Leung*
    Anthony K. L. Leung
    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, United States
    McKusick-Nathans Department of Genetics Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
    Department of Oncology  and  Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States
    *Email: [email protected]
Cite this: ACS Chem. Biol. 2022, 17, 1, 17–23
Publication Date (Web):December 14, 2021
https://doi.org/10.1021/acschembio.1c00721
Copyright © 2021 American Chemical Society
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Abstract

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Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen that identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib inhibits SARS-CoV-2 and MERS-CoV Mac1 but not the closest human homologue, MacroD2. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for the screening of large compound libraries to identify improved macrodomain inhibitors and to explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

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Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.1c00721.

  • Materials and Methods; Supplementary Figures S1–S8: Sequence alignment of coronavirus macrodomains, enzymatic and structural comparisons of SARS-CoV-2 Mac1 and MacroD2, optimizing assay parameters for drug screening, evaluation of pilot screen hits, surface plasmon resonance traces, molecular docking of dasatinib with SARS-CoV-2 Mac1, structure-based sequence alignment of MacroD2 with other human macrodomains, and comparison of dasatinib with hits identified in previously published screens (PDF)

  • Supplementary Data File 1: Pilot screen raw data (XLSX)

  • Supplementary Data File 2: Conservation of SARS-CoV-2 genomes (XLSX)

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Cited By

This article is cited by 12 publications.

  1. Sarah Wazir, Tomi A. O. Parviainen, Jessica J. Pfannenstiel, Men Thi Hoai Duong, Daniel Cluff, Sven T. Sowa, Albert Galera-Prat, Dana Ferraris, Mirko M. Maksimainen, Anthony R. Fehr, Juha P. Heiskanen, Lari Lehtiö. Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target. Journal of Medicinal Chemistry 2024, 67 (8) , 6519-6536. https://doi.org/10.1021/acs.jmedchem.3c02451
  2. Kyuto Tashiro, Sven Wijngaarden, Jugal Mohapatra, Johannes G. M. Rack, Ivan Ahel, Dmitri V. Filippov, Glen Liszczak. Chemoenzymatic and Synthetic Approaches To Investigate Aspartate- and Glutamate-ADP-Ribosylation. Journal of the American Chemical Society 2023, 145 (25) , 14000-14009. https://doi.org/10.1021/jacs.3c03771
  3. Ananya Anmangandla, Sadhan Jana, Kewen Peng, Shamar D. Wallace, Saket R. Bagde, Bryon S. Drown, Jiashu Xu, Paul J. Hergenrother, J. Christopher Fromme, Hening Lin. A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain. ACS Chemical Biology 2023, 18 (5) , 1200-1207. https://doi.org/10.1021/acschembio.3c00092
  4. Giuliana Catara, Rocco Caggiano, Luca Palazzo. The DarT/DarG Toxin–Antitoxin ADP-Ribosylation System as a Novel Target for a Rational Design of Innovative Antimicrobial Strategies. Pathogens 2023, 12 (2) , 240. https://doi.org/10.3390/pathogens12020240
  5. Marion Schuller, Tryfon Zarganes-Tzitzikas, James Bennett, Stephane De Cesco, Daren Fearon, Frank von Delft, Oleg Fedorov, Paul E. Brennan, Ivan Ahel. Discovery and Development Strategies for SARS-CoV-2 NSP3 Macrodomain Inhibitors. Pathogens 2023, 12 (2) , 324. https://doi.org/10.3390/pathogens12020324
  6. Johannes Gregor Matthias Rack, Ivan Ahel. A Simple Method to Study ADP-Ribosylation Reversal: From Function to Drug Discovery. 2023, 111-132. https://doi.org/10.1007/978-1-0716-2891-1_8
  7. Arash Keshavarzi Arshadi, Milad Salem, Arash Firouzbakht, Jiann Shiun Yuan. MolData, a molecular benchmark for disease and target based machine learning. Journal of Cheminformatics 2022, 14 (1) https://doi.org/10.1186/s13321-022-00590-y
  8. Maria D. Politi, Angelo Gallo, Georgios Bouras, Maria Birkou, Bruno Canard, Bruno Coutard, Georgios A. Spyroulias. 1H, 13C, 15N backbone resonance assignment of apo and ADP-ribose bound forms of the macro domain of Hepatitis E virus through solution NMR spectroscopy. Biomolecular NMR Assignments 2022, 330 https://doi.org/10.1007/s12104-022-10111-5
  9. Lavinia M. Sherrill, Elva E. Joya, AnnMarie Walker, Anuradha Roy, Yousef M. Alhammad, Moriama Atobatele, Sarah Wazir, George Abbas, Patrick Keane, Junlin Zhuo, Anthony K.L. Leung, David K. Johnson, Lari Lehtiö, Anthony R. Fehr, Dana Ferraris. Design, synthesis and evaluation of inhibitors of the SARS-CoV-2 nsp3 macrodomain. Bioorganic & Medicinal Chemistry 2022, 67 , 116788. https://doi.org/10.1016/j.bmc.2022.116788
  10. Anu Roy, Yousef M. Alhammad, Peter McDonald, David K. Johnson, Junlin Zhuo, Sarah Wazir, Dana Ferraris, Lari Lehtiö, Anthony K.L. Leung, Anthony R. Fehr. Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening. Antiviral Research 2022, 203 , 105344. https://doi.org/10.1016/j.antiviral.2022.105344
  11. Bernhard Lüscher, Maud Verheirstraeten, Sarah Krieg, Patricia Korn. Intracellular mono-ADP-ribosyltransferases at the host–virus interphase. Cellular and Molecular Life Sciences 2022, 79 (6) https://doi.org/10.1007/s00018-022-04290-6
  12. Anthony K. L. Leung, Diane E. Griffin, Jürgen Bosch, Anthony R. Fehr. The Conserved Macrodomain Is a Potential Therapeutic Target for Coronaviruses and Alphaviruses. Pathogens 2022, 11 (1) , 94. https://doi.org/10.3390/pathogens11010094

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