K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

Current address Biological Sciences Division Pacific Northwest National Laboratory 902 Battelle Blvd Richland WA 99353

Department of Biochemistry and Biophysics Texas A and M University 301 Old Main Drive College Station Texas 77843

Department of Chemistry Texas A and M University 301 Old Main Drive College Station Texas 77843

Department of Medicine Division of Infectious Diseases and Global Public Health University of California San Diego La Jolla CA 92037 USA

Department of Microbiology and Immunology University of Texas Medical Branch 3000 University Boulevard Galveston Texas 77755 1001

Institute for Antiviral Research Department of Animal Dairy and Veterinary Sciences 5600 Old Main Hill Utah State University Logan Utah 84322

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic 16610 Prague Czech Republic

Selva Therapeutics Department of Animal Dairy and Veterinary Sciences 5600 Old Main Hill Utah State University Logan Utah 84322

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA

ACS Chem Biol. 2021 Apr 16;16(4):642-650. doi: 10.1021/acschembio.0c00875. PubMed

Zobrazit více v PubMed

Zhou P, et al. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579(7798):270–273. PubMed PMC

Morse JS, Lalonde T, Xu S, & Liu WR (2020) Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV. ChemBioChem 21(5):730–738. PubMed PMC

Dong E, Du H, & Gardner L (2020) An interactive web-based dashboard to track COVID-19 in real time. The Lancet Infectious Diseases 20(5):533–534. PubMed PMC

New York Times (2020) Coronavirus in the U.S.: Latest Map and Case Count.

Pillaiyar T, Meenakshisundaram S, & Manickam M (2020) Recent discovery and development of inhibitors targeting coronaviruses. Drug Discovery Today 25(4):668–688. PubMed PMC

Guy RK, DiPaola RS, Romanelli F, & Dutch RE (2020) Rapid repurposing of drugs for COVID-19. Science 368(6493):829. PubMed

Rota PA, et al. (2003) Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. Science 300(5624):1394. PubMed

Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, & Fouchier RAM (2012) Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. New England Journal of Medicine 367(19):1814–1820. PubMed

Lu R, et al. (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395(10224):565–574. PubMed PMC

Zhang L, et al. (2020) Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science 368(6489):409. PubMed PMC

Báez-Santos YM, St John SE, & Mesecar AD (2015) The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds. Antiviral research 115:21–38. PubMed PMC

Li L, Li G, Chen J, Liang X, & Li Y (2020) Comparative genomic analysis revealed specific mutation pattern between human coronavirus SARS-CoV-2 and Bat-SARSr-CoV RaTG13. bioRxiv:2020.2002.2027.969006. PubMed PMC

Heald-Sargent T & Gallagher T (2012) Ready, set, fuse! The coronavirus spike protein and acquisition of fusion competence. Viruses 4(4):557–580. PubMed PMC

Shang J, et al. (2020) Cell entry mechanisms of SARS-CoV-2. Proceedings of the National Academy of Sciences 117(21):11727. PubMed PMC

Ou X, et al. (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature Communications 11(1):1620. PubMed PMC

Belouzard S, Chu VC, & Whittaker GR (2009) Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proceedings of the National Academy of Sciences 106(14):5871. PubMed PMC

Millet JK & Whittaker GR (2014) Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein. Proceedings of the National Academy of Sciences 111(42):15214. PubMed PMC

Hoffmann M, et al. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181(2):271–280.e278. PubMed PMC

Huang Y, Yang C, Xu X-f, Xu W, & Liu S-w (2020) Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacologica Sinica 41(9):1141–1149 PubMed PMC

Simmons G, et al. (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proceedings of the National Academy of Sciences of the United States of America 102(33):11876. PubMed PMC

Zhou Y, et al. (2015) Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research 116:76–84. PubMed PMC

Bosch BJ, Bartelink W, & Rottier PJM (2008) Cathepsin L Functionally Cleaves the Severe Acute Respiratory Syndrome Coronavirus Class I Fusion Protein Upstream of Rather than Adjacent to the Fusion Peptide. Journal of Virology 82(17):8887. PubMed PMC

Riva L, et al. (2020) Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing. Nature 586(7827):113–119. PubMed PMC

Palmer JT, Rasnick D, Klaus JL, & Bromme D (1995) Vinyl Sulfones as Mechanism-Based Cysteine Protease Inhibitors. Journal of Medicinal Chemistry 38(17):3193–3196. PubMed

Engel JC, Doyle PS, Hsieh I, & McKerrow JH (1998) Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection. J Exp Med 188(4):725–734. PubMed PMC

Yang PY, Wang M, He CY, Yao SQ. (2012) Proteomic profiling and potential cellular target identification of K11777, a clinical cysteine protease inhibitor, in Trypanosoma brucei. Chem Commun 48(6):835–837. PubMed

Doyle PS, Zhou YM, Engel JC, & McKerrow JH (2007) A Cysteine Protease Inhibitor Cures Chagas Disease in an Immunodeficient-Mouse Model of Infection. Antimicrobial Agents and Chemotherapy 51(11):3932. PubMed PMC

Barr SC, et al. (2005) A cysteine protease inhibitor protects dogs from cardiac damage during infection by Trypanosoma cruzi. Antimicrobial agents and chemotherapy 49(12):5160–5161. PubMed PMC

Abdulla M-H, Lim K-C, Sajid M, McKerrow JH, & Caffrey CR (2007) Schistosomiasis Mansoni: Novel Chemotherapy Using a Cysteine Protease Inhibitor. PLOS Medicine 4(1):e14. PubMed PMC

Ndao M, et al. (2013) A Cysteine Protease Inhibitor Rescues Mice from a Lethal Cryptosporidium parvum Infection. Antimicrobial Agents and Chemotherapy 57(12):6063. PubMed PMC

McKerrow JH (2018) Update on drug development targeting parasite cysteine proteases. PLoS neglected tropical diseases 12(8):e0005850–e0005850. PubMed PMC

Vuong W, et al. (2020) Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication. Nature Communications 11(1):4282. PubMed PMC

Schneider CA, Rasband WS, & Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9(7):671–675. PubMed PMC

Thoms M, et al. (2020) Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369(6508):1249. PubMed PMC

Cox J, et al. (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Molecular & cellular proteomics : MCP 13(9):2513–2526. PubMed PMC

Jaimes JA, André NM, Chappie JS, Millet JK, & Whittaker GR (2020) Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. Journal of molecular biology 432(10):3309–3325. PubMed PMC

Walls AC, et al. (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181(2):281–292.e286. PubMed PMC

Tang T, et al. (2020) Proteolytic activation of the SARS-CoV-2 spike S1/S2 site: a re-evaluation of furin cleavage. bioRxiv:2020.2010.2004.325522.

Park J-E, et al. (2016) Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism. Proceedings of the National Academy of Sciences 113(43):12262. PubMed PMC