Structure and Inhibition of the SARS-CoV-2 Main Protease Reveal Strategy for Developing Dual Inhibitors Against M and Cathepsin L

Overview

Journal Sci Adv

Specialties Biology
Science

Date 2020 Nov 7

PMID 33158912

Citations 225

Authors

Michael Dominic Sacco

Chunlong Ma

Panagiotis Lagarias

Ang Gao

Julia Alma Townsend

Xiangzhi Meng

Peter Dube

XiuJun Zhang

Yanmei Hu

Naoya Kitamura

Brett Hurst

Bart Tarbet

Michael Thomas Marty

Antonios Kolocouris

Yan Xiang

Yu Chen

Jun Wang

Affiliations

Soon will be listed here.

Abstract

The main protease (M) of SARS-CoV-2 is a key antiviral drug target. While most M inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several M inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of M in complex with calpain inhibitors II and XII and three analogs of The structure of M with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.

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References

Eastman R, Roth J, Brimacombe K, Simeonov A, Shen M, Patnaik S . Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19. ACS Cent Sci. 2020; 6(5):672-683. PMC: 7202249. DOI: 10.1021/acscentsci.0c00489. View

Pedersen N, Kim Y, Liu H, Galasiti Kankanamalage A, Eckstrand C, Groutas W . Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg. 2017; 20(4):378-392. PMC: 5871025. DOI: 10.1177/1098612X17729626. View

Yang S, Chen S, Hsu M, Wu J, Tseng C, Liu Y . Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor. J Med Chem. 2006; 49(16):4971-80. DOI: 10.1021/jm0603926. View

Sheahan T, Sims A, Zhou S, Graham R, Pruijssers A, Agostini M . An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci Transl Med. 2020; 12(541). PMC: 7164393. DOI: 10.1126/scitranslmed.abb5883. View

Dai W, Zhang B, Jiang X, Su H, Li J, Zhao Y . Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science. 2020; 368(6497):1331-1335. PMC: 7179937. DOI: 10.1126/science.abb4489. View

Vagin A, Teplyakov A . Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 1):22-5. DOI: 10.1107/S0907444909042589. View

Mandadapu S, Gunnam M, Tiew K, Uy R, Prior A, Alliston K . Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors. Bioorg Med Chem Lett. 2012; 23(1):62-5. PMC: 3586229. DOI: 10.1016/j.bmcl.2012.11.026. View

Kim Y, Liu H, Galasiti Kankanamalage A, Weerasekara S, Hua D, Groutas W . Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor. PLoS Pathog. 2016; 12(3):e1005531. PMC: 4814111. DOI: 10.1371/journal.ppat.1005531. View

Xia S, Liu M, Wang C, Xu W, Lan Q, Feng S . Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res. 2020; 30(4):343-355. PMC: 7104723. DOI: 10.1038/s41422-020-0305-x. View

10.

Zhu L, George S, Schmidt M, Al-Gharabli S, Rademann J, Hilgenfeld R . Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease. Antiviral Res. 2011; 92(2):204-12. PMC: 7114241. DOI: 10.1016/j.antiviral.2011.08.001. View

11.

Murshudov G, Skubak P, Lebedev A, Pannu N, Steiner R, Nicholls R . REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011; 67(Pt 4):355-67. PMC: 3069751. DOI: 10.1107/S0907444911001314. View

12.

Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng E . UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13):1605-12. DOI: 10.1002/jcc.20084. View

13.

Verschueren K, Pumpor K, Anemuller S, Chen S, Mesters J, Hilgenfeld R . A structural view of the inactivation of the SARS coronavirus main proteinase by benzotriazole esters. Chem Biol. 2008; 15(6):597-606. PMC: 7110992. DOI: 10.1016/j.chembiol.2008.04.011. View

14.

Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M . Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020; 30(3):269-271. PMC: 7054408. DOI: 10.1038/s41422-020-0282-0. View

15.

Ma C, Sacco M, Hurst B, Townsend J, Hu Y, Szeto T . Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell Res. 2020; 30(8):678-692. PMC: 7294525. DOI: 10.1038/s41422-020-0356-z. View

16.

Liu C, Zhou Q, Li Y, Garner L, Watkins S, Carter L . Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Cent Sci. 2020; 6(3):315-331. PMC: 10467574. DOI: 10.1021/acscentsci.0c00272. View

17.

Sheahan T, Sims A, Graham R, Menachery V, Gralinski L, Case J . Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med. 2017; 9(396). PMC: 5567817. DOI: 10.1126/scitranslmed.aal3653. View

18.

Marty M, Baldwin A, Marklund E, Hochberg G, Benesch J, Robinson C . Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal Chem. 2015; 87(8):4370-6. PMC: 4594776. DOI: 10.1021/acs.analchem.5b00140. View

19.

Muramatsu T, Takemoto C, Kim Y, Wang H, Nishii W, Terada T . SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity. Proc Natl Acad Sci U S A. 2016; 113(46):12997-13002. PMC: 5135343. DOI: 10.1073/pnas.1601327113. View

20.

Agostini M, Andres E, Sims A, Graham R, Sheahan T, Lu X . Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio. 2018; 9(2). PMC: 5844999. DOI: 10.1128/mBio.00221-18. View