CaltechAUTHORS
Published May 7, 2021 | Version Supplemental Material + Submitted + Published
Journal Article Open

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

Creators

  • 1. ROR icon Center for Free-Electron Laser Science
  • 2. ROR icon Bernhard Nocht Institute for Tropical Medicine
  • 3. ROR icon Diamond Light Source
  • 4. ROR icon Universität Hamburg
  • 5. ROR icon European X-Ray Free-Electron Laser
  • 6. ROR icon Heinrich-Pette-Institute
  • 7. ROR icon Max Planck Institute for Molecular Genetics
  • 8. ROR icon Max Planck Institute for the Structure and Dynamics of Matter
  • 9. ROR icon University of California, Irvine
  • 10. ROR icon Deutsches Elektronen-Synchrotron DESY
  • 11. ROR icon Hamburg University of Technology
  • 12. ROR icon California Institute of Technology
  • 13. ROR icon Fraunhofer Society
  • 14. ROR icon European Molecular Biology Laboratory
  • 15. ROR icon University of Lübeck
  • 16. ROR icon Helmholtz-Zentrum Berlin für Materialien und Energie
  • 17. ROR icon Jožef Stefan Institute
  • 18. ROR icon Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins
  • 19. ROR icon Leibniz University Hannover
  • 20. ROR icon University of Greifswald

Abstract

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Additional Information

© 2021 American Association for the Advancement of Science. Received 20 November 2020; accepted 29 March 2021; Published online April 2, 2021. We acknowledge Deutsches Elektronen-Synchrotron (DESY, Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at PETRA III at beamline P11. Further MX data were collected at beamline P13 and P14 operated by EMBL. We thank the DESY machine group, in particular Mario Wunderlich, Kim Heuck, Arne Brinkmann, Olaf Goldbeck, Jürgen Haar, Torsten Schulz, Gunnar Priebe, Maximilian Holz, Björn Lemcke, Klaus Knaack, Oliver Seebauer, Philipp Willanzheimer, Rolf Jonas, and Nicole Engling. We thank Thomas Dietrich, Simon Geile, Filip Guicking, Heshmat Noei, and Tim Pakendorf from DESY, and Bianca Di Fabrizio and Sebastian Kühn from BNITM for assistance. This research was supported in part through the Maxwell computational resources operated at DESY. We acknowledge the use of the XBI biological sample preparation laboratory at European XFEL, enabled by the XBI User Consortium. We acknowledge financial support from the EXSCALATE4CoV EU-H2020 Emergency Project (101003551), the Cluster of Excellence "Advanced Imaging of Matter" of the Deutsche Forschungsgemeinschaft (DFG) - EXC 2056 - project ID 390715994, the Helmholtz Association Impulse and Networking funds (projects ExNet-0002 and InternLabs-0011 "HIR3X"), the Federal Ministry of Education and Research (BMBF) via projects 05K16GUA, 05K19GU4, 05K20BI1, 05K20FL1, 16GW0277 and 031B0405D), and the Joachim-Herz-Stiftung Hamburg (project Infecto-Physics). CE and MR acknowledge financial support from grant-No. HIDSS-0002 DASHH (Data Science in Hamburg - HELMHOLTZ Graduate School for the Structure of Matter). RC is supported by DFG grants INST 187/621-1 and INST 187/686-1. DT is supported by the Slovenian Research Agency (ARRS; research program P1-0048, Infrastructural program IO-0048). BS was supported by an Exploration Grant from the Boehringer Ingelheim Foundation. The Heinrich Pette Institute, Leibniz Institute for Experimental Virology was supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health. CU and BK were supported by EU Horizon 2020 ERC StG-2017 759661, BMBF RTK Struktur 01KI20391, BMBF Visavix 05K16BH1 and the Leibniz Association SAW-2014-HPI-4 grant. Author Contributions: SeG, PR, YFG, WB, PG, ARB, RC, DT, AZ, HNC, ARP, CB, AM designed research. SeG, PR, TJL, WH, HNC, ARP, CB, AM wrote manuscript. SeG, PR, JL, FHMK, SM, WB, ID, BS, HGie, BNB, MB, PLX, NW, HA, NU, SF, BAF, MS, HB, JK, GEPM, ARM, FG, VH, PF, MW, ECS, PM, HT, TB participated in sample preparation. PR performed crystallization experiments. SeG, PR, JL, TJL, OY, SS, AT, MGr, HF, FT, MGa, YG, CFL, SA, AP, GB, DVS, GP, TRS, IB, SP performed X-ray data collection. TJL, HGin, DO, OY, LG, MD, TAW, FS, CR, DM, JZD, IK, CS, RS, HUH, DCFM contributed to X-ray data management. SeG, PR, JL, TJL, HGin, FHMK, WE, DO, AH, VS, JH, JM, JB, JW, CF, MSW, AC, DT, WH, AM performed X-ray data analysis. KL, BK, CU, RC performed and analyzed MS experiments. YFG, BEP, StG performed and analyzed antiviral activity assays. PG, BE, MK, MGA, SN, CG, LZ, XS, KK, AU, JL, RH performed and analyzed ligand binding studies and protein activity assays. CE, JPZ, MR performed computational binding studies. Competing Interests: MR is stakeholder of BioSolveIT GmbH, licensor of the software HYDE. Data and materials availability: The coordinates and structure factors for all described crystal structures of SARS-CoV-2 Mpro in complex with compounds are deposited in the PDB with accession codes 6YNQ, 6YVF, 7A1U, 7ABU, 7ADW, 7AF0, 7AGA, 7AHA, 7AK4, 7AKU, 7AMJ, 7ANS, 7AOL, 7AP6, 7APH, 7AQE, 7AQI, 7AQJ, 7AR5, 7AR6, 7ARF, 7AVD, 7AWR, 7AWS, 7AWU, 7AWW, 7AX6, 7AXM, 7AXO, 7AY7, 7B83 and 7NEV. Code used in this analysis has been previously published (10). The code for forcing adherence to the Wilson distribution is included in the Vagabond refinement package (https://vagabond.hginn.co.uk/) under a GPLv3 license. Compounds from the Fraunhofer IME Repurposing collection were obtained from the Fraunhofer Institute for Molecular Biology and Applied Ecology under a Material Transfer Agreement. Compounds from the Safe-in-man Library were kindly provided by Dompé Farmaceutici S.p.A. Other materials are available from SeG or AM upon request. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

Attached Files

Published - 642.full.pdf

Submitted - 2020.11.12.378422v2.full.pdf

Supplemental Material - abf7945-Guenther-SM.pdf

Supplemental Material - abf7945_Guenther_Reproducibility-Checklist.pdf

Supplemental Material - abf7945_Guenther_Table-S1.xlsx

Supplemental Material - abf7945_Guenther_Table-S3.pdf

Supplemental Material - abf7945_Guenther_Table-S4.xlsx

Supplemental Material - abf7945_Guenther_Table-S7.xlsx

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Additional details

Additional titles

Alternative title
Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease
Alternative title
Inhibition of SARS-CoV-2 main protease by allosteric drug-binding

Identifiers

PMCID
PMC8224385
Eprint ID
106665
Resolver ID
CaltechAUTHORS:20201113-100151878

Related works

Describes
10.1101/2020.11.12.378422 (DOI)

Funding

European Union
EXSCALATE4CoV
European Union
101003551
Deutsche Forschungsgemeinschaft (DFG)
EXC 2056
Deutsche Forschungsgemeinschaft (DFG)
390715994
Helmholtz-Gemeinschaft Deutscher Forschungszentren (HGF)
ExNet-0002
Helmholtz-Gemeinschaft Deutscher Forschungszentren (HGF)
InternLabs-0011 "HIR3X"
Bundesministerium für Bildung und Forschung (BMBF)
05K16GUA
Bundesministerium für Bildung und Forschung (BMBF)
05K19GU4
Bundesministerium für Bildung und Forschung (BMBF)
05K20BI1
Bundesministerium für Bildung und Forschung (BMBF)
05K20FL1
Bundesministerium für Bildung und Forschung (BMBF)
16GW0277
Bundesministerium für Bildung und Forschung (BMBF)
031B0405D
Joachim-Herz-Stiftung Hamburg
Helmholtz Graduate School for the Structure of Matter
HIDSS-0002
Deutsche Forschungsgemeinschaft (DFG)
187/621-1
Deutsche Forschungsgemeinschaft (DFG)
187/686-1
Slovenian Research Agency
P1-0048
Slovenian Research Agency
IO-0048
Boehringer Ingelheim Foundation
City of Hamburg
Bundesgesundheitsministerium
European Research Council (ERC)
759661
Bundesministerium für Bildung und Forschung (BMBF)
01KI2039
Bundesministerium für Bildung und Forschung (BMBF)
05K16BH1
Leibniz Association
SAW-2014-HPI-4

Dates

Created
2020-11-13
Created from EPrint's datestamp field
Updated
2021-08-05
Created from EPrint's last_modified field

Caltech Custom Metadata

Caltech groups
COVID-19