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Citation: Mengxin Zhou, Yutong Han, Mengxia Li, Gang Ye, Guiqing Peng. Two Inhibitors Against the 3C-Like Proteases of Swine Coronavirus and Feline Coronavirus [J].VIROLOGICA SINICA, 2021, 36(6) : 1421-1430.  http://dx.doi.org/10.1007/s12250-021-00415-6

Two Inhibitors Against the 3C-Like Proteases of Swine Coronavirus and Feline Coronavirus

  • Corresponding author: Guiqing Peng, penggq@mail.hzau.edu.cn, ORCID: 0000-0001-5419-2499
  • Received Date: 19 February 2021
    Accepted Date: 06 May 2021
    Published Date: 06 July 2021
    Available online: 01 December 2021
  • Coronaviruses (CoVs) are important human and animal pathogens that cause respiratory and gastrointestinal diseases. Porcine epidemic diarrhoea (PED), characterized by severe diarrhoea and vomiting in pigs, is a highly lethal disease caused by porcine epidemic diarrhoea virus (PEDV) and causes substantial losses in the swine industry worldwide. However, currently available commercial drugs have not shown great therapeutic effects. In this study, a fluorescence resonance energy transfer (FRET)-based assay was applied to screen a library containing 1, 590 compounds and identified two compounds, 3-(aminocarbonyl)-1-phenylpyridinium and 2, 3-dichloronaphthoquinone, that target the 3C-like protease (3CLpro) of PEDV. These compounds are of low molecular weight (MW) and greatly inhibited the activity of this enzyme (IC50 values were obtained in this study). Furthermore, these compounds exhibited antiviral capacity against another member of the CoV family, feline infectious peritonitis virus (FIPV). Here, the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified (with EC50 values) and cell viability assays were performed. The results of putative molecular docking models indicate that these compounds, labeled compound 1 and compound 2, contact the conserved active sites (Cys144, Glu165, Gln191) of 3CLpro via hydrogen bonds. These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.


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    1. Chen F, Zhu Y, Wu M, Ku X, Ye S, Li Z, Guo X, He Q (2015) Comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus. Viruses 7: 5525–5538
        doi: 10.3390/v7102891

    2. Choudhury B, Dastjerdi A, Doyle N, Frossard JP, Steinbach F (2016) From the field to the lab-an European view on the global spread of PEDV. Virus Res 226: 40–49
        doi: 10.1016/j.virusres.2016.09.003

    3. Congreve M, Carr R, Murray C, Jhoti H (2003) A 'Rule of Three' for fragment-based lead discovery? Drug Discov Today 8: 876–877
        doi: 10.1016/S1359-6446(03)02831-9

    4. Cui J, Li F, Shi ZL (2019) Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17: 181–192
        doi: 10.1038/s41579-018-0118-9

    5. Gerdts V, Zakhartchouk A (2017) Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Vet Microbiol 206: 45–51
        doi: 10.1016/j.vetmic.2016.11.029

    6. Ghosh AK, Xi K, Ratia K, Santarsiero BD, Fu W, Harcourt BH, Rota PA, Baker SC, Johnson ME, Mesecar AD (2005) Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors. J Med Chem 48: 6767–6771
        doi: 10.1021/jm050548m

    7. Grum-Tokars V, Ratia K, Begaye A, Baker SC, Mesecar AD (2008) Evaluating the 3C-like protease activity of SARS-Coronavirus: recommendations for standardized assays for drug discovery. Virus Res 133: 63–73
        doi: 10.1016/j.virusres.2007.02.015

    8. Guo YR, Cao QD, Hong ZS, Tan YY, Chen SD, Jin HJ, Tan KS, Wang DY, Yan Y (2020) The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status. Mil Med Res 7: 11
        doi: 10.1186/s40779-020-00240-0

    9. Hofmann M, Wyler R (1988) Propagation of the virus of porcine epidemic diarrhea in cell culture. J Clin Microbiol 26: 2235–2239
        doi: 10.1128/jcm.26.11.2235-2239.1988

    10. Jo S, Kim S, Shin DH, Kim MS (2020) Inhibition of SARS-CoV 3CL protease by flavonoids. J Enzyme Inhib Med Chem 35: 145–151
        doi: 10.1080/14756366.2019.1690480

    11. Jung K, Saif LJ (2015) Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J 204: 134–143
        doi: 10.1016/j.tvjl.2015.02.017

    12. Kankanamalage ACG, Kim Y, Damalanka VC, Rathnayake AD, Fehr AR, Mehzabeen N, Battaile KP, Lovell S, Lushington GH, Perlman S, Chang KO, Groutas WC (2018) Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element. Eur J Med Chem 150: 334–346
        doi: 10.1016/j.ejmech.2018.03.004

    13. Kim Y, Mandadapu SR, Groutas WC, Chang KO (2013) Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease. Antiviral Res 97: 161–168
        doi: 10.1016/j.antiviral.2012.11.005

    14. Kim Y, Liu H, Kankanamalage ACG, Weerasekara S, Hua DH, Groutas WC, Chang KO, Pedersen NC (2016) Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog 12: e1005531
        doi: 10.1371/journal.ppat.1005531

    15. Kipar A, Meli ML (2014) Feline infectious peritonitis: still an enigma? Vet Pathol 51: 505–526
        doi: 10.1177/0300985814522077

    16. Kumar V, Shin JS, Shie JJ, Ku KB, Kim C, Go YY, Huang KF, Kim M, Liang PH (2017) Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CL(Pro) inhibitors. Antiviral Res 141: 101–106
        doi: 10.1016/j.antiviral.2017.02.007

    17. Langel SN, Paim FC, Lager KM, Vlasova AN, Saif LJ (2016) Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV): historical and current concepts. Virus Res 226: 93–107
        doi: 10.1016/j.virusres.2016.05.016

    18. Li W, Li H, Liu Y, Pan Y, Deng F, Song Y, Tang X, He Q (2012) New variants of porcine epidemic diarrhea virus, China, 2011. Emerg Infect Dis 18: 1350–1353
        doi: 10.3201/eid1803.120002

    19. Mokaya J, McNaughton AL, Hadley MJ, Beloukas A, Geretti AM, Goedhals D, Matthews PC (2018) A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: a call for urgent action. PLoS Negl Trop Dis 12: e0006629
        doi: 10.1371/journal.pntd.0006629

    20. Omoto S, Speranzini V, Hashimoto T, Noshi T, Yamaguchi H, Kawai M, Kawaguchi K, Uehara T, Shishido T, Naito A, Cusack S (2018) Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Sci Rep 8: 9633
        doi: 10.1038/s41598-018-27890-4

    21. Pedersen NC (2009) A review of feline infectious peritonitis virus infection: 1963–2008. J Feline Med Surg 11: 225–258
        doi: 10.1016/j.jfms.2008.09.008

    22. Pedersen NC (2014) An update on feline infectious peritonitis: diagnostics and therapeutics. Vet J 201: 133–141
        doi: 10.1016/j.tvjl.2014.04.016

    23. Pensaert MB, de Bouck P (1978) A new coronavirus-like particle associated with diarrhea in swine. Arch Virol 58: 243–247
        doi: 10.1007/BF01317606

    24. Pensaert MB, Martelli P (2016) Porcine epidemic diarrhea: a retrospect from Europe and matters of debate. Virus Res 226: 1–6
        doi: 10.1016/j.virusres.2016.05.030

    25. Perera KD, Kankanamalage ACG, Rathnayake AD, Honeyfield A, Groutas W, Chang KO, Kim Y (2018) Protease inhibitors broadly effective against feline, ferret and mink coronaviruses. Antiviral Res 160: 79–86
        doi: 10.1016/j.antiviral.2018.10.015

    26. Perera KD, Rathnayake AD, Liu H, Pedersen NC, Groutas WC, Chang KO, Kim Y (2019) Characterization of amino acid substitutions in feline coronavirus 3C-like protease from a cat with feline infectious peritonitis treated with a protease inhibitor. Vet Microbiol 237: 108398
        doi: 10.1016/j.vetmic.2019.108398

    27. Pfefferle S, Schöpf J, Kögl M, Friedel CC, Müller MA, Carbajo-Lozoya J, Stellberger T, von Dall'Armi E, Herzog P, Kallies S, Niemeyer D, Ditt V, Kuri T, Züst R, Pumpor K, Hilgenfeld R, Schwarz F, Zimmer R, Steffen I, Weber F, Thiel V, Herrler G, Thiel HJ, Schwegmann-Wessels C, Pöhlmann S, Haas J, Drosten C, von Brunn A (2011) The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7: e1002331
        doi: 10.1371/journal.ppat.1002331

    28. Pillaiyar T, Manickam M, Namasivayam V, Hayashi Y, Jung SH (2016) An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors: peptidomimetics and small molecule chemotherapy. J Med Chem 59: 6595–6628
        doi: 10.1021/acs.jmedchem.5b01461

    29. Ryu YB, Jeong HJ, Kim JH, Kim YM, Park JY, Kim D, Nguyen TT, Park SJ, Chang JS, Park KH, Rho MC, Lee WS (2010) Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition. Bioorg Med Chem 18: 7940–7947
        doi: 10.1016/j.bmc.2010.09.035

    30. Schoeman D, Fielding BC (2019) Coronavirus envelope protein: current knowledge. Virol J 16: 69
        doi: 10.1186/s12985-019-1182-0

    31. Shi Y, Li Y, Lei Y, Ye G, Shen Z, Sun L, Luo R, Wang D, Fu ZF, Xiao S, Peng G (2016) A dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp11. J Virol 90: 4579–4592
        doi: 10.1128/JVI.03065-15

    32. Shie JJ, Fang JM, Kuo TH, Kuo CJ, Liang PH, Huang HJ, Wu YT, Jan JT, Cheng YS, Wong CH (2005) Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha, beta-unsaturated esters. Bioorg Med Chem 13: 5240–5252
        doi: 10.1016/j.bmc.2005.05.065

    33. St John SE, Tomar S, Stauffer SR, Mesecar AD (2015) Targeting zoonotic viruses: Structure-based inhibition of the 3C-like protease from bat coronavirus HKU4–The likely reservoir host to the human coronavirus that causes Middle East Respiratory Syndrome (MERS). Bioorg Med Chem 23: 6036–6048
        doi: 10.1016/j.bmc.2015.06.039

    34. Stobart CC, Lee AS, Lu X, Denison MR (2012) Temperature-sensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3CLpro) define residues involved in long-distance communication and regulation of protease activity. J Virol 86: 4801–4810
        doi: 10.1128/JVI.06754-11

    35. Theerawatanasirikul S, Kuo CJ, Phetcharat N, Lekcharoensuk P (2020) In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus. Antiviral Res 174: 104697
        doi: 10.1016/j.antiviral.2019.104697

    36. Wang D, Fang L, Xiao S (2016) Porcine epidemic diarrhea in China. Virus Res 226: 7–13
        doi: 10.1016/j.virusres.2016.05.026

    37. Wang Q, Vlasova AN, Kenney SP, Saif LJ (2019) Emerging and re-emerging coronaviruses in pigs. Curr Opin Virol 34: 39–49
        doi: 10.1016/j.coviro.2018.12.001

    38. Wood EN (1977) An apparently new syndrome of porcine epidemic diarrhoea. Vet Rec 100: 243–244
        doi: 10.1136/vr.100.12.243

    39. Xie W, Ao C, Yang Y, Liu Y, Liang R, Zeng Z, Ye G, Xiao S, Fu ZF, Dong W, Peng G (2019) Two critical N-terminal epitopes of the nucleocapsid protein contribute to the cross-reactivity between porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus. J Gen Virol 100: 206–216
        doi: 10.1099/jgv.0.001216

    40. Yang S, Chen SJ, Hsu MF, Wu JD, Tseng CT, Liu YF, Chen HC, Kuo CW, Wu CS, Chang LW, Chen WC, Liao SY, Chang TY, Hung HH, Shr HL, Liu CY, Huang YA, Chang LY, Hsu JC, Peters CJ, Wang AH, Hsu MC (2006) Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor. J Med Chem 49: 4971–4980
        doi: 10.1021/jm0603926

    41. Yang DQ, Ge FF, Ju HB, Wang J, Liu J, Ning K, Liu PH, Zhou JP, Sun QY (2014) Whole-genome analysis of porcine epidemic diarrhea virus (PEDV) from eastern China. Arch Virol 159: 2777–2785
        doi: 10.1007/s00705-014-2102-7

    42. Ye G, Deng F, Shen Z, Luo R, Zhao L, Xiao S, Fu ZF, Peng G (2016) Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease. Virology 494: 225–235
        doi: 10.1016/j.virol.2016.04.018

    43. Ye G, Wang X, Tong X, Shi Y, Fu ZF, Peng G (2020) Structural basis for inhibiting porcine epidemic diarrhea virus replication with the 3C-like protease inhibitor GC376. Viruses 12: 240
        doi: 10.3390/v12020240

    44. Zhu L, George S, Schmidt MF, Al-Gharabli SI, Rademann J, Hilgenfeld R (2011) Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease. Antiviral Res 92: 204–212
        doi: 10.1016/j.antiviral.2011.08.001

    45. Ziebuhr J (2005) The coronavirus replicase. Curr Top Microbiol Immunol 287: 57–94

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    Two Inhibitors Against the 3C-Like Proteases of Swine Coronavirus and Feline Coronavirus

      Corresponding author: Guiqing Peng, penggq@mail.hzau.edu.cn
    • 1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China
    • 2. Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China

    Abstract: 

    Coronaviruses (CoVs) are important human and animal pathogens that cause respiratory and gastrointestinal diseases. Porcine epidemic diarrhoea (PED), characterized by severe diarrhoea and vomiting in pigs, is a highly lethal disease caused by porcine epidemic diarrhoea virus (PEDV) and causes substantial losses in the swine industry worldwide. However, currently available commercial drugs have not shown great therapeutic effects. In this study, a fluorescence resonance energy transfer (FRET)-based assay was applied to screen a library containing 1, 590 compounds and identified two compounds, 3-(aminocarbonyl)-1-phenylpyridinium and 2, 3-dichloronaphthoquinone, that target the 3C-like protease (3CLpro) of PEDV. These compounds are of low molecular weight (MW) and greatly inhibited the activity of this enzyme (IC50 values were obtained in this study). Furthermore, these compounds exhibited antiviral capacity against another member of the CoV family, feline infectious peritonitis virus (FIPV). Here, the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified (with EC50 values) and cell viability assays were performed. The results of putative molecular docking models indicate that these compounds, labeled compound 1 and compound 2, contact the conserved active sites (Cys144, Glu165, Gln191) of 3CLpro via hydrogen bonds. These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.