Citation: Yue Wang, Huimin Huang, Dongliang Li, Chenxu Zhao, Shuai Li, Panpan Qin, Yaqin Li, Xia Yang, Wenjuan Du, Wentao Li, Yongtao Li. Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization .VIROLOGICA SINICA, 2023, 38(2) : 296-308.  http://dx.doi.org/10.1016/j.virs.2023.01.008

Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization

  • Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there remain no effective drugs against PEDV infection. In this study, we utilized a recombinant PEDV expressing renilla luciferase (PEDV-Rluc) to screen potential anti-PEDV agents from an FDA-approved drug library in Vero cells. Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc. Among them, niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index. It can efficiently inhibit viral RNA synthesis, protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner. Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection. Furthermore, niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells. In addition, a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro. Taken together, these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

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    1. Belouzard, S., Millet, J.K., Licitra, B.N.,Whittaker, G.R., 2012. Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses, 4, 1011-1033.

    2. Braga, L., Ali, H., Secco, I., Chiavacci, E., Neves, G., Goldhill, D., Penn, R., Jimenez-Guardeño, J.M., Ortega-Prieto, A.M., Bussani, R., Cannatà, A., Rizzari, G., Collesi, C., Schneider, E., Arosio, D., Shah, A.M., Barclay, W.S., Malim, M.H., Burrone, J.,Giacca, M., 2021. Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia. Nature, 594, 88-93.

    3. Burkard, C., Verheije, M.H., Wicht, O., Van Kasteren, S.I., Van Kuppeveld, F.J., Haagmans, B.L., Pelkmans, L., Rottier, P.J., Bosch, B.J.,De Haan, C.A., 2014. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner. PLoS Pathog, 10, e1004502.

    4. Cao, L., Ge, X., Gao, Y., Ren, Y., Ren, X.,Li, G., 2015. Porcine epidemic diarrhea virus infection induces NF-κB activation through the TLR2, TLR3 and TLR9 pathways in porcine intestinal epithelial cells. J Gen Virol, 96, 1757-1767.

    5. Chang, Z., Wang, Y., Zhou, X.,Long, J.E., 2018. STAT3 roles in viral infection:antiviral or proviral? Future Virol, 13, 557-574.

    6. Chen, W., Mook, R.A., Jr., Premont, R.T.,Wang, J., 2018. Niclosamide:Beyond an antihelminthic drug. Cell Signal, 41, 89-96.

    7. De Almeida, L., Da Silva, A.L.N., Rodrigues, T.S., Oliveira, S., Ishimoto, A.Y., Seribelli, A.A., Becerra, A., Andrade, W.A., Ataide, M.A., Caetano, C.C.S., De Sá, K.S.G., Pelisson, N., Martins, R.B., De Paula Souza, J., Arruda, E., Batah, S.S., Castro, R., Frantz, F.G., Cunha, F.Q., Cunha, T.M., Fabro, A.T., Cunha, L.D., Louzada-Junior, P., De Oliveira, R.D.R.,Zamboni, D.S., 2022. Identification of immunomodulatory drugs that inhibit multiple inflammasomes and impair SARS-CoV-2 infection. Sci Adv, 8, eabo5400.

    8. Dong, S., Yu, R., Wang, X., Chen, B., Si, F., Zhou, J., Xie, C., Li, Z.,Zhang, D., 2022. Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus In Vitro and In Vivo. Viruses, 14.

    9. Eaton, A.F., Merkulova, M.,Brown, D., 2021. The H(+)-ATPase (V-ATPase):from proton pump to signaling complex in health and disease. Am J Physiol Cell Physiol, 320, C392-c414.

    10. Gassen, N.C., Papies, J., Bajaj, T., Emanuel, J., Dethloff, F., Chua, R.L., Trimpert, J., Heinemann, N., Niemeyer, C., Weege, F., Hönzke, K., Aschman, T., Heinz, D.E., Weckmann, K., Ebert, T., Zellner, A., Lennarz, M., Wyler, E., Schroeder, S., Richter, A., Niemeyer, D., Hoffmann, K., Meyer, T.F., Heppner, F.L., Corman, V.M., Landthaler, M., Hocke, A.C., Morkel, M., Osterrieder, N., Conrad, C., Eils, R., Radbruch, H., Giavalisco, P., Drosten, C.,Müller, M.A., 2021. SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals. Nat Commun, 12, 3818.

    11. Gerdts, V.,Zakhartchouk, A., 2017. Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Vet Microbiol, 206, 45-51.

    12. Harrison, S.C., 2008. Viral membrane fusion. Nat Struct Mol Biol, 15, 690-698.

    13. Hemmat, N., Asadzadeh, Z., Ahangar, N.K., Alemohammad, H., Najafzadeh, B., Derakhshani, A., Baghbanzadeh, A., Baghi, H.B., Javadrashid, D., Najafi, S., Ar Gouilh, M.,Baradaran, B., 2021. The roles of signaling pathways in SARS-CoV-2 infection; lessons learned from SARS-CoV and MERS-CoV. Arch Virol, 166, 675-696.

    14. Huang, H., Li, Y., Li, D., Wang, L., Jiao, W., Bai, Y.,Zhang, G., 2022. The tyrosine phosphatase PTPN14 inhibits the activation of STAT3 in PEDV infected Vero cells. Vet Microbiol, 267, 109391.

    15. Huang, Y.W., Dickerman, A.W., Piñeyro, P., Li, L., Fang, L., Kiehne, R., Opriessnig, T.,Meng, X.J., 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio, 4, e00737-00713.

    16. Huynh, J., Chand, A., Gough, D.,Ernst, M., 2019. Therapeutically exploiting STAT3 activity in cancer-using tissue repair as a road map. Nat Rev Cancer, 19, 82-96.

    17. Icho, S., Rujas, E., Muthuraman, K., Tam, J., Liang, H., Landreth, S., Liao, M., Falzarano, D., Julien, J.P.,Melnyk, R.A., 2022. Dual Inhibition of Vacuolar-ATPase and TMPRSS2 Is Required for Complete Blockade of SARS-CoV-2 Entry into Cells. Antimicrob Agents Chemother, 66, e0043922.

    18. Jeon, S., Ko, M., Lee, J., Choi, I., Byun, S.Y., Park, S., Shum, D.,Kim, S., 2020. Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved Drugs. Antimicrob Agents Chemother, 64.

    19. Jitobaom, K., Boonarkart, C., Manopwisedjaroen, S., Punyadee, N., Borwornpinyo, S., Thitithanyanont, A., Avirutnan, P., Auewarakul, P.J.B.P.,Toxicology, 2022. Synergistic anti-SARS-CoV-2 activity of repurposed anti-parasitic drug combinations. 23, 1-13.

    20. Jung, E., Nam, S., Oh, H., Jun, S., Ro, H.J., Kim, B., Kim, M.,Go, Y.Y., 2019. Neutralization of Acidic Intracellular Vesicles by Niclosamide Inhibits Multiple Steps of the Dengue Virus Life Cycle In Vitro. Sci Rep, 9, 8682.

    21. Jung, K., Saif, L.J.,Wang, Q., 2020. Porcine epidemic diarrhea virus (PEDV):An update on etiology, transmission, pathogenesis, and prevention and control. Virus Res, 286, 198045.

    22. Jurgeit, A., Mcdowell, R., Moese, S., Meldrum, E., Schwendener, R.,Greber, U.F., 2012. Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects. PLoS Pathog, 8, e1002976.

    23. Kadri, H., Lambourne, O.A.,Mehellou, Y., 2018. Niclosamide, a Drug with Many (Re)purposes. ChemMedChem, 13, 1088-1091.

    24. Kao, J.C., Huangfu, W.C., Tsai, T.T., Ho, M.R., Jhan, M.K., Shen, T.J., Tseng, P.C., Wang, Y.T.,Lin, C.F., 2018. The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR. PLoS Negl Trop Dis, 12, e0006715.

    25. Kumar, N., Sharma, S., Kumar, R., Tripathi, B.N., Barua, S., Ly, H.,Rouse, B.T., 2020. Host-Directed Antiviral Therapy. Clin Microbiol Rev, 33.

    26. 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.

    27. Li, X., Sun, J., Prinz, R.A., Liu, X.,Xu, X., 2020. Inhibition of porcine epidemic diarrhea virus (PEDV) replication by A77 1726 through targeting JAK and Src tyrosine kinases. Virology, 551, 75-83.

    28. Li, Y., Li, P., He, Q., Zhang, R., Li, Y., Kamar, N., Peppelenbosch, M.P., De Man, R.A., Wang, L.,Pan, Q., 2022. Niclosamide inhibits hepatitis E virus through suppression of NF-kappaB signalling. Antiviral Res, 197, 105228.

    29. Li, Y., Li, P.K., Roberts, M.J., Arend, R.C., Samant, R.S.,Buchsbaum, D.J., 2014. Multi-targeted therapy of cancer by niclosamide:A new application for an old drug. Cancer Lett, 349, 8-14.

    30. Lin, H., Li, B., Liu, M., Zhou, H., He, K.,Fan, H., 2020. Nonstructural protein 6 of porcine epidemic diarrhea virus induces autophagy to promote viral replication via the PI3K/Akt/mTOR axis. Vet Microbiol, 244, 108684.

    31. Liu, B., Palmfeldt, J., Lin, L., Colaço, A., Clemmensen, K.K.B., Huang, J., Xu, F., Liu, X., Maeda, K., Luo, Y.,Jäättelä, M., 2018. STAT3 associates with vacuolar H(+)-ATPase and regulates cytosolic and lysosomal pH. Cell Res, 28, 996-1012.

    32. Liu, C., Ma, Y., Yang, Y., Zheng, Y., Shang, J., Zhou, Y., Jiang, S., Du, L., Li, J.,Li, F., 2016. Cell Entry of Porcine Epidemic Diarrhea Coronavirus Is Activated by Lysosomal Proteases. J Biol Chem, 291, 24779-24786.

    33. Matsuyama, S., Ujike, M., Morikawa, S., Tashiro, M.,Taguchi, F., 2005. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. Proc Natl Acad Sci U S A, 102, 12543-12547.

    34. Matsuyama, T., Kubli, S.P., Yoshinaga, S.K., Pfeffer, K.,Mak, T.W., 2020. An aberrant STAT pathway is central to COVID-19. Cell Death Differ, 27, 3209-3225.

    35. Mazzon, M.,Marsh, M., 2019. Targeting viral entry as a strategy for broad-spectrum antivirals. F1000Res, 8.

    36. Mazzon, M., Ortega-Prieto, A.M., Imrie, D., Luft, C., Hess, L., Czieso, S., Grove, J., Skelton, J.K., Farleigh, L., Bugert, J.J., Wright, E., Temperton, N., Angell, R., Oxenford, S., Jacobs, M., Ketteler, R., Dorner, M.,Marsh, M., 2019. Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry. Viruses, 11.

    37. Mccartney, E.M., Helbig, K.J., Narayana, S.K., Eyre, N.S., Aloia, A.L.,Beard, M.R., 2013. Signal transducer and activator of transcription 3 is a proviral host factor for hepatitis C virus. Hepatology, 58, 1558-1568.

    38. Medicine, U.S.N.L.O. 2022. Niclosamide clinical trials[Online]. Available:https://clinicaltrials.gov/ct2/show/NCT04542434[Accessed 20 January 2022].

    39. Millet, J.K.,Whittaker, G.R., 2015. Host cell proteases:Critical determinants of coronavirus tropism and pathogenesis. Virus Res, 202, 120-134.

    40. Okemoto, K., Wagner, B., Meisen, H., Haseley, A., Kaur, B.,Chiocca, E.A., 2013. STAT3 activation promotes oncolytic HSV1 replication in glioma cells. PLoS One, 8, e71932.

    41. Park, J.E., Cruz, D.J.,Shin, H.J., 2014. Clathrin- and serine proteases-dependent uptake of porcine epidemic diarrhea virus into Vero cells. Virus Res, 191, 21-29.

    42. Prabhakara, C., Godbole, R., Sil, P., Jahnavi, S., Zanten, T., Sheth, D., Subhash, N., Chandra, A., Nuthakki, V.K.,Puthiyapurayil, T.P.J.C.S.H.L., 2020. Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway. BioRxiv.

    43. Qi, X., Cao, Y., Wu, S., Wu, Z.,Bao, W., 2021. miR-129a-3p Inhibits PEDV Replication by Targeting the EDA-Mediated NF-κB Pathway in IPEC-J2 Cells. Int J Mol Sci, 22.

    44. Qin, H.R., Kim, H.J., Kim, J.Y., Hurt, E.M., Klarmann, G.J., Kawasaki, B.T., Duhagon Serrat, M.A.,Farrar, W.L., 2008. Activation of signal transducer and activator of transcription 3 through a phosphomimetic serine 727 promotes prostate tumorigenesis independent of tyrosine 705 phosphorylation. Cancer Res, 68, 7736-7741.

    45. Shi, W., Fan, W., Bai, J., Tang, Y., Wang, L., Jiang, Y., Tang, L., Liu, M., Cui, W., Xu, Y.,Li, Y., 2017. TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells. Viruses, 9.

    46. Singh, S., Weiss, A., Goodman, J., Fisk, M., Kulkarni, S., Lu, I., Gray, J., Smith, R., Sommer, M.,Cheriyan, J., 2022. Niclosamide-A promising treatment for COVID-19. Br J Pharmacol, 179, 3250-3267.

    47. Song, D., Moon, H.,Kang, B., 2015. Porcine epidemic diarrhea:a review of current epidemiology and available vaccines. Clin Exp Vaccine Res, 4, 166-176.

    48. Stevenson, G.W., Hoang, H., Schwartz, K.J., Burrough, E.R., Sun, D., Madson, D., Cooper, V.L., Pillatzki, A., Gauger, P., Schmitt, B.J., Koster, L.G., Killian, M.L.,Yoon, K.J., 2013. Emergence of Porcine epidemic diarrhea virus in the United States:clinical signs, lesions, and viral genomic sequences. J Vet Diagn Invest, 25, 649-654.

    49. Wang, Y.,Stark, G.R., 2018. A new STAT3 function:pH regulation. Cell Res, 28, 1045.

    50. Wang, Y.M., Lu, J.W., Lin, C.C., Chin, Y.F., Wu, T.Y., Lin, L.I., Lai, Z.Z., Kuo, S.C.,Ho, Y.J., 2016. Antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission. Antiviral Res, 135, 81-90.

    51. Wei, X., She, G., Wu, T., Xue, C.,Cao, Y., 2020. PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway. Vet Res, 51, 10.

    52. Weiss, A., Touret, F., Baronti, C., Gilles, M., Hoen, B., Nougairède, A., De Lamballerie, X.,Sommer, M.O.A., 2021. Niclosamide shows strong antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency against the Alpha (B.1.1.7), Beta (B.1.351) and Delta variant (B.1.617.2). PLoS One, 16, e0260958.

    53. Wermuth, C.G., 2004. Selective optimization of side activities:another way for drug discovery. J Med Chem, 47, 1303-1314.

    54. Wu, C.J., Jan, J.T., Chen, C.M., Hsieh, H.P., Hwang, D.R., Liu, H.W., Liu, C.Y., Huang, H.W., Chen, S.C., Hong, C.F., Lin, R.K., Chao, Y.S.,Hsu, J.T., 2004. Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide. Antimicrob Agents Chemother, 48, 2693-2696.

    55. Xu, J., Shi, P.Y., Li, H.,Zhou, J., 2020. Broad Spectrum Antiviral Agent Niclosamide and Its Therapeutic Potential. ACS Infect Dis, 6, 909-915.

    56. Yang, L., Xu, J., Guo, L., Guo, T., Zhang, L., Feng, L., Chen, H.,Wang, Y., 2018. Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon. J Virol, 92.

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    Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization

      Corresponding author: Wentao Li, wentao@mail.hzau.edu.cn
      Corresponding author: Yongtao Li, yongtaole@126.com
    • a. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China;
    • b. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China;
    • c. Faculty of Veterinary Medicine, Utrecht University, Utrecht, 3584 CL, the Netherlands;
    • d. Hubei Hongshan Laboratory, Wuhan, 430070, China

    Abstract: Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there remain no effective drugs against PEDV infection. In this study, we utilized a recombinant PEDV expressing renilla luciferase (PEDV-Rluc) to screen potential anti-PEDV agents from an FDA-approved drug library in Vero cells. Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc. Among them, niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index. It can efficiently inhibit viral RNA synthesis, protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner. Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection. Furthermore, niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells. In addition, a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro. Taken together, these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

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