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

Günther, Sebastian and Reinke, Patrick Y. A. and Fernández-García, Yaiza and Lieske, Julia and Lane, Thomas J. and Ginn, Helen M. and Koua, Faisal H. M. and Ehrt, Christiane and Ewert, Wiebke and Oberthuer, Dominik and Yefanov, Oleksandr and Meier, Susanne and Lorenzen, Kristina and Krichel, Boris and Kopicki, Janine-Denise and Gelisio, Luca and Brehm, Wolfgang and Dunkel, Ilona and Seychell, Brandon and Gieseler, Henry and Norton-Baker, Brenna and Escudero-Pérez, Beatriz and Domaracky, Martin and Saouane, Sofiane and Tolstikova, Aleksandra and White, Thomas A. and Hänle, Anna and Groessler, Michael and Fleckenstein, Holger and Trost, Fabian and Galchenkova, Marina and Gevorkov, Yaroslav and Li, Chufeng and Awel, Salah and Peck, Ariana and Barthelmess, Miriam and Schlünzen, Frank and Lourdu Xavier, Paulraj and Werner, Nadine and Andaleeb, Hina and Ullah, Najeeb and Falke, Sven and Srinivasan, Vasundara and Alves França, Bruno and Schwinzer, Martin and Brognaro, Hévila and Rogers, Cromarte and Melo, Diogo and Zaitsev-Doyle, Joanna J. and Knoska, Juraj and Peña-Murillo, Gisel E. and Rahmani Mashhour, Aida and Hennicke, Vincent and Fischer, Pontus and Hakanpää, Johanna and Meyer, Jan and Gribbon, Philip and Ellinger, Bernhard and Kuzikov, Maria and Wolf, Markus and Beccari, Andrea R. and Borenkov, Gleb and von Stetten, David and Pompidor, Guillaume and Bento, Isabel and Panneerselvam, Saravanan and Karpics, Ivars and Schneider, Thomas R. and Garcia-Alai, Maria Marta and Niebling, Stephan and Günther, Christian and Schmidt, Christina and Schubert, Robin and Han, Huijong and Boger, Juliane and Monteiro, Diana C. F. and Zhang, Linlin and Sun, Xinyuanyuan and Pletzer-Zelgert, Jonathan and Wollenhaupt, Jan and Feiler, Christian G. and Weiss, Manfred S. and Schulz, Eike-Christian and Mehrabi, Pedram and Karničar, Katarina and Usenik, Aleksandra and Loboda, Jure and Tidow, Henning and Chari, Ashwin and Hilgenfeld, Rolf and Uetrecht, Charlotte and Cox, Russell and Zaliani, Andrea and Beck, Tobias and Rarey, Matthias and Günther, Stephan and Turk, Dusan and Hinrichs, Winfried and Chapman, Henry N. and Pearson, Arwen R. and Betzel, Christian and Meents, Alke (2021) X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease. Science, 372 (6542). pp. 642-646. ISSN 0036-8075. PMCID PMC8224385. doi:10.1126/science.abf7945.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription Materials CentralArticle Paper
Günther, Sebastian0000-0002-7329-6653
Reinke, Patrick Y. A.0000-0002-7354-0839
Fernández-García, Yaiza0000-0002-9592-2141
Ginn, Helen M.0000-0002-7894-4939
Koua, Faisal H. M.0000-0001-8371-9587
Ehrt, Christiane0000-0003-1428-0042
Yefanov, Oleksandr0000-0001-8676-0091
Kopicki, Janine-Denise0000-0001-9806-5352
Gelisio, Luca0000-0001-7832-6201
Escudero-Pérez, Beatriz0000-0003-3655-0525
Domaracky, Martin0000-0003-4624-023X
Saouane, Sofiane0000-0002-7260-9021
Tolstikova, Aleksandra0000-0003-0653-2123
Fleckenstein, Holger0000-0002-7216-6494
Trost, Fabian0000-0001-5628-598X
Gevorkov, Yaroslav0000-0001-9273-7648
Li, Chufeng0000-0002-2997-0903
Awel, Salah0000-0002-9470-5911
Peck, Ariana0000-0002-5940-3897
Barthelmess, Miriam0000-0002-1874-0117
Lourdu Xavier, Paulraj0000-0001-5132-2999
Falke, Sven0000-0003-3409-1791
Srinivasan, Vasundara0000-0002-8895-5885
Alves França, Bruno0000-0001-6008-6000
Knoska, Juraj0000-0001-7258-6256
Peña-Murillo, Gisel E.0000-0001-7842-4987
Hennicke, Vincent0000-0002-2997-2722
Fischer, Pontus0000-0002-5543-6018
Meyer, Jan0000-0002-6498-9976
Gribbon, Philip0000-0001-7655-2459
Ellinger, Bernhard0000-0001-5703-3901
von Stetten, David0000-0001-7906-9788
Bento, Isabel0000-0003-3801-4929
Niebling, Stephan0000-0001-6582-5984
Schubert, Robin0000-0002-6213-2872
Han, Huijong0000-0002-1197-3014
Monteiro, Diana C. F.0000-0002-6318-7306
Wollenhaupt, Jan0000-0002-3418-5213
Feiler, Christian G.0000-0002-9214-5770
Weiss, Manfred S.0000-0002-2362-7047
Usenik, Aleksandra0000-0003-2366-2886
Loboda, Jure0000-0002-9601-3484
Tidow, Henning0000-0002-4702-9332
Chari, Ashwin0000-0001-6220-9828
Hilgenfeld, Rolf0000-0001-8850-2977
Uetrecht, Charlotte0000-0002-1991-7922
Cox, Russell0000-0002-1844-0157
Zaliani, Andrea0000-0002-1740-8390
Rarey, Matthias0000-0002-9553-6531
Günther, Stephan0000-0002-6562-0230
Turk, Dusan0000-0003-0205-6609
Hinrichs, Winfried0000-0002-0435-4565
Chapman, Henry N.0000-0002-4655-1743
Pearson, Arwen R.0000-0001-8499-7490
Betzel, Christian0000-0002-3879-5019
Meents, Alke0000-0001-6078-4095
Alternate Title:Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease, Inhibition of SARS-CoV-2 main protease by allosteric drug-binding
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 ( 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 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.
Funding AgencyGrant Number
European UnionEXSCALATE4CoV
European Union101003551
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 HamburgUNSPECIFIED
Helmholtz Graduate School for the Structure of MatterHIDSS-0002
Deutsche Forschungsgemeinschaft (DFG)187/621-1
Deutsche Forschungsgemeinschaft (DFG)187/686-1
Slovenian Research AgencyP1-0048
Slovenian Research AgencyIO-0048
Boehringer Ingelheim FoundationUNSPECIFIED
City of HamburgUNSPECIFIED
European Research Council (ERC)759661
Bundesministerium für Bildung und Forschung (BMBF)01KI2039
Bundesministerium für Bildung und Forschung (BMBF)05K16BH1
Leibniz AssociationSAW-2014-HPI-4
Issue or Number:6542
PubMed Central ID:PMC8224385
Record Number:CaltechAUTHORS:20201113-100151878
Persistent URL:
Official Citation:X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease. S. Günther et al., Science 372 (6542), 642-646; DOI: 10.1126/science.abf7945 (2021)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106665
Deposited By: George Porter
Deposited On:13 Nov 2020 19:27
Last Modified:05 Aug 2021 16:38

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