Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (M pro , also called 3CL pro ) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 M pro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 M pro . The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.

Shown for reading. Its publisher allows quoting it, not republishing it.

References 42

1. 1.
   A pneumonia outbreak associated with a new coronavirus of probable bat origin · 2020

   No notice
2. 2.
   A new coronavirus associated with human respiratory disease in China · 2020

   Corrected
3. 3.
   The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 · 2020

   No notice
4. 4.

   Anand K., Ziebuhr J., Wadhwani P., Mesters J. R., Hilgenfeld R., Coronavirus main proteinase (3CLpro) structure: Basis for design of anti-SARS drugs. Science 300, 1763–1767 (2003 ).

   Not in our library10.1126/science.1085658

5. 5.

   Hilgenfeld R., From SARS to MERS: Crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 281, 4085–4096 (2014 ).

   Not in our library10.1111/febs.12936

6. 6.

   Zhang L., Lin D., Kusov Y., Nian Y., Ma Q., Wang J., von Brunn A., Leyssen P., Lanko K., Neyts J., de Wilde A., Snijder E. J., Liu H., Hilgenfeld R., α-Ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: Structure-based design, synthesis, and activity assessment. J. Med. Chem. acs.jmedchem.9b01828 (2020 ).

   Not in our library10.1021/acs.jmedchem.9b01828

7. 7.

   Tan J., Verschueren K. H. G., Anand K., Shen J., Yang M., Xu Y., Rao Z., Bigalke J., Heisen B., Mesters J. R., Chen K., Shen X., Jiang H., Hilgenfeld R., pH-dependent conformational flexibility of the SARS-CoV main proteinase (Mpro) dimer: Molecular dynamics simulations and multiple X-ray structure analyses. J. Mol. Biol. 354, 25–40 (2005 ).

   Not in our library10.1016/j.jmb.2005.09.012

8. 8.

   Shi J., Song J., The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain. FEBS J. 273, 1035–1045 (2006 ).

   Not in our library10.1111/j.1742-4658.2006.05130.x

9. 9.

   Anand K., Palm G. J., Mesters J. R., Siddell S. G., Ziebuhr J., Hilgenfeld R., Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. EMBO J. 21, 3213–3224 (2002 ).

   Not in our library10.1093/emboj/cdf327

10. 10.
    Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain. · 2014

    Not checked
11. 11.

    Kratochwil N. A., Huber W., Müller F., Kansy M., Gerber P. R., Predicting plasma protein binding of drugs: A new approach. Biochem. Pharmacol. 64, 1355–1374 (2002 ).

    Not in our library10.1016/s0006-2952(02)01074-2

12. 12.

    Yang H., Yang M., Ding Y., Liu Y., Lou Z., Zhou Z., Sun L., Mo L., Ye S., Pang H., Gao G. F., Anand K., Bartlam M., Hilgenfeld R., Rao Z., The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc. Natl. Acad. Sci. U.S.A. 100, 13190–13195 (2003 ).

    Not in our library10.1073/pnas.1835675100

13. 13.
    Only One Protomer Is Active in the Dimer of SARS 3C-like Proteinase · 2006

    No notice
14. 14.

    Zhu L., George S., Schmidt M. F., Al-Gharabli S. I., Rademann J., Hilgenfeld R., Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease. Antiviral Res. 92, 204–212 (2011 ).

    Not in our library10.1016/j.antiviral.2011.08.001

15. 15.

    Tan J., George S., Kusov Y., Perbandt M., Anemüller S., Mesters J. R., Norder H., Coutard B., Lacroix C., Leyssen P., Neyts J., Hilgenfeld R., 3C protease of enterovirus 68: Structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses. J. Virol. 87, 4339–4351 (2013 ).

    Not in our library10.1128/jvi.01123-12

16. 16.

    Dragovich P. S., Zhou R., Skalitzky D. J., Fuhrman S. A., Patick A. K., Ford C. E., Meador J. W. 3rd, Worland S. T., Solid-phase synthesis of irreversible human rhinovirus 3C protease inhibitors. Part 1: Optimization of tripeptides incorporating N-terminal amides. Bioorg. Med. Chem. 7, 589–598 (1999 ).

    Not in our library10.1016/s0968-0896(99)00005-x

17. 17.

    L. Zhang, D. Lin, R. Hilgenfeld, Crystal structure of the complex resulting from the reaction between the SARS-CoV main protease and tert-butyl (1-((S)-3-cyclohexyl-1-(((S)-4-(cyclopropylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl) amino)-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate, PDB ID 6Y7M (2020).

    Not in our library10.2210/pdb6y7m/pdb

18. 18.

    L. Zhu, R. Hilgenfeld, Crystal structure of SARS coronavirus main protease complexed with an alpha, beta-unsaturated ethyl ester inhibitor SG85, PDB ID 3TNT (2012).

    Not in our library10.2210/pdb3tnt/pdb

19. 19.

    Kusov Y., Tan J., Alvarez E., Enjuanes L., Hilgenfeld R., A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication-transcription complex. Virology 484, 313–322 (2015 ).

    Not in our library10.1016/j.virol.2015.06.016

20. 20.

    Xue X., Yang H., Shen W., Zhao Q., Li J., Yang K., Chen C., Jin Y., Bartlam M., Rao Z., Production of authentic SARS-CoV Mpro with enhanced activity: Application as a novel tag-cleavage endopeptidase for protein overproduction. J. Mol. Biol. 366, 965–975 (2007 ).

    Not in our library10.1016/j.jmb.2006.11.073

21. 21.
    Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin · 2012

    No notice
22. 22.

    Krug M., Weiss M. S., Heinemann U., Mueller U., XDSAPP: A graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 45, 568–572 (2012 ).

    Not in our library10.1107/s0021889812011715

23. 23.

    Evans P., Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006 ).

    Not in our library10.1107/s0907444905036693

24. 24.
    An introduction to data reduction: space-group determination, scaling and intensity statistics · 2011

    No notice
25. 25.
    Overview of the CCP 4 suite and current developments · 2011

    No notice
26. 26.

    Vagin A., Teplyakov A., Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010 ).

    Not in our library10.1107/s0907444909042589

27. 27.
    JLigand : a graphical tool for the CCP 4 template-restraint library · 2012

    No notice
28. 28.
    Features and development of Coot · 2010

    No notice
29. 29.
    REFMAC 5 for the refinement of macromolecular crystal structures · 2011

    No notice
30. 30.

    Liu Y., Kati W., Chen C. M., Tripathi R., Molla A., Kohlbrenner W., Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal. Biochem. 267, 331–335 (1999 ).

    Not in our library10.1006/abio.1998.3014

31. 31.

    P. H. Brown, A. Balbo, P. Schuck, Characterizing protein-protein interactions by sedimentation velocity analytical ultracentrifugation. Curr. Protoc. Immunol. Chapter 18, Unit 18.15 (2008).

    Not in our library10.1002/0471142735.im1815s81

32. 32.

    Schuck P., Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000 ).

    Not in our library10.1016/s0006-3495(00)76713-0

33. 33.

    M. T. Laue, B. D. Shah, T. M. Rigdeway, S. L. Pelletier, in Analytical Ultracentrifugation in Biochemistry and Polymer Science, S. Harding, A. Rowe, J. Horton, Eds. (Royal Society of Chemistry, 1992), pp. 90–125.

    Not in our library

34. 34.

    Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E., UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004 ).

    Not in our library10.1002/jcc.20084

35. 35.

    Tian Q., Nayyar N. K., Babu S., Chen L., Tao J., Lee S., Tibbetts A., Moran T., Liou J., Guo M., Kennedy T. P., An efficient synthesis of a key intermediate for the preparation of the rhinovirus protease inhibitor AG7088 via asymmetric dianionic cyanomethylation of N-Boc-L-(+)-glutamic acid dimethyl ester. Tetrahedron Lett. 42, 6807–6809 (2001 ).

    Not in our library10.1016/s0040-4039(01)01416-2

36. 36.
    Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR · 2020

    Expression of concern
37. 37.

    Zhang Y., Huo M., Zhou J., Xie S., PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 99, 306–314 (2010 ).

    Not in our library10.1016/j.cmpb.2010.01.007

38. 38.

    Hsu W. C., Chang H. C., Chou C. Y., Tsai P. J., Lin P. I., Chang G. G., Critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease. J. Biol. Chem. 280, 22741–22748 (2005 ).

    Not in our library10.1074/jbc.m502556200

39. 39.

    Bräutigam C. A., Calculations and publication-quality illustrations for analytical ultracentrifugation data. Methods Enzymol. 562, 109–133 (2015 ).

    Not in our library10.1016/bs.mie.2015.05.001

40. 40.

    Weiss M. S., Hilgenfeld R., On the use of the merging R factor as a quality indicator for X-ray data. J. Appl. Crystallogr. 30, 203–205 (1997 ).

    Not in our library10.1107/s0021889897003907

41. 41.

    Karplus P. A., Diederichs K., Linking crystallographic model and data quality. Science 336, 1030–1033 (2012 ).

    Not in our library10.1126/science.1218231

42. 42.
    MolProbity : all-atom structure validation for macromolecular crystallography · 2009

    No notice

Cited by papers we hold 66

• The main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII · 2025
  Not checked
• Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors · 2023
  No notice
• SARS-CoV-2 3CL pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376 · 2023
  No notice
• Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors · 2023
  No notice
• In silico analysis of SARS-CoV-2 proteins as targets for clinically available drugs · 2022
  Not checked
• A Computer-Aided Approach for the Discovery of D-Peptides as Inhibitors of SARS-CoV-2 Main Protease · 2022
  Not checked
• Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 Mpro/3CLpro in Living Cells · 2022
  Not checked
• Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system · 2022
  No notice
• Structural biology of SARS-CoV-2: open the door for novel therapies · 2022
  No notice
• Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance · 2022
  No notice
• Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2 · 2022
  No notice
• Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay · 2022
  No notice
• Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19 · 2022
  No notice
• SARS-CoV-2 NSP5 and N protein counteract the RIG-I signaling pathway by suppressing the formation of stress granules · 2022
  No notice
• The SARS‐CoV‐2 main protease (M pro ): Structure, function, and emerging therapies for COVID‐19 · 2022
  No notice
• Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease · 2021
  No notice
• Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19 · 2021
  No notice
• Potential inhibitors of SARS-CoV-2 (COVID 19) proteases PLpro and Mpro/ 3CLpro: molecular docking and simulation studies of three pertinent medicinal plant natural components · 2021
  Not checked
• Biflavonoid as potential 3-chymotrypsin-like protease (3CLpro) inhibitor of SARS-Coronavirus · 2021
  Not checked
• X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease · 2021
  No notice

and 46 more

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.