In Silico and In Vitro Antiviral Activity Evaluation of Prodigiosin from Serratia marcescens Against Enterovirus 71
Keywords:
Cytotoxicity, FireDock, HFMD, MTT, prodigiosin, virucidalAbstract
Prodigiosin, a red linear tripyrrole pigment found in Serratia marcescens, is one such naturally occurring compound that has gained wide attention owing to its numerous biological activities, including antibacterial, antifungal, antimalarial, anticancer, and immunosuppressive properties. This study was conducted to evaluate the possible antiviral activity of prodigiosin against Enterovirus 71, a causative agent of hand, foot, and mouth disease (HFMD). Preliminary studies were done in silico by analyzing the interaction of prodigiosin with amino acid residues of five EV71-target proteins. Interaction refinement analysis with FireDock revealed that 2C helicase (-48.01 kcal/moL) has the most negative global energy, followed by capsid (-36.52 kcal/moL), 3C protease (-34.16 kcal/moL), 3D RNA polymerase (-30.93 kcal/moL) and 2A protease (-20.61 kcal/moL). These values are indicative of the interaction strength. Prodigiosin was shown to form chemical bonds with specific amino acid residues in capsid (Gln-30, Asn-223), 2A protease (Trp-33, Trp-142), 2C helicase (Tyr-150, His-151, Gln-169, Ser-212), 3C protease (Glu-50), and 3D RNA polymerase (Ala-239, Tyr-237). To investigate further, prodigiosin was extracted from S. marcescens using a methanolic extraction method. In vitro studies revealed that prodigiosin, with an IC50 value of 0.5112 μg/mL, reduced virus titers by 0.17 log (32.39%) in 30 min and 0.19 log (35.43%) in 60 min. The findings suggest that prodigiosin has antiviral activity with an intermediate inhibitory effect against EV71. As a result of this research, new biological activities of prodigiosin have been identified.
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Abdullah, N.H. 2022. Situasi semasa kejadian penyakit tangan, kaki dan mulut (HFMD) di Malaysia [WWW Document]. URL https://kpkesihatan.com/2022/06/14/kenyataan-akhbar-kpk-14-jun-2022-situasi-semasa-kejadian-penyakit-tangan-kaki-dan-mulut-hfmd-di-malaysia-me-23-2022/ (accessed 9.11.2022).
Andrusier, N., Nussinov, R. & Wolfson, H.J. 2007. FireDock: Fast interaction refinement in molecular docking. Proteins: Structure, Function and Genetics, 69(1): 139–159. DOI: https://doi.org/10.1002/prot.21495
Arita, M., Wakita, T. & Shimizu, H. 2008. Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity. Journal of General Virology, 89(10): 2518–2530. DOI: https://doi.org/10.1099/vir.0.2008/002915-0
Bonvin, A.M. 2006. Flexible protein-protein docking. Current Opinion in Structural Biology, 16(2): 194–200. DOI: https://doi.org/10.1016/j.sbi.2006.02.002
Borowski, P., Niebuhr, A., Schmitz, H., Hosmane, R.S., Bretner, M., Siwecka, M.A. & Kulikowski, T. 2002. NTPase/helicase of Flaviviridae: inhibitors and inhibition of the enzyme. Acta Biochimica Polonica, 49(3): 597–614. DOI: https://doi.org/10.18388/abp.2002_3769
Cai, Q., Yameen, M., Liu, W., Gao, Z., Li, Y., Peng, X., Cai, Y., Wu, C., Zheng, Q., Li, J. & Lin, T. 2013. Conformational plasticity of the 2A proteinase from Enterovirus 71. Journal of Virology, 87(13): 7348–7356. DOI: https://doi.org/10.1128/JVI.03541-12
CDC. 2022. Hand, foot, and mouth disease [WWW Document]. URL https://wwwnc.cdc.gov/travel/diseases/hand-foot-and-mouth-disease (accessed 9.11.2022).
Chen, C., Wang, Y., Shan, C., Sun, Y., Xu, P., Zhou, H., Yang, C., Shi, P.Y., Rao, Z., Zhang, B. & Lou, Z. 2013. Crystal structure of Enterovirus 71 RNA-dependent RNA polymerase complexed with its protein primer VPg: implication for a trans mechanism of VPg uridylylation. Journal of Virology, 87(10): 5755–5768. DOI: https://doi.org/10.1128/JVI.02733-12
Choi, H.J., Lim, C.H., Song, J.H., Baek, S.H. & Kwon, D.H. 2009. Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses. Phytomedicine, 16(1): 35–39. DOI: https://doi.org/10.1016/j.phymed.2008.10.012
Chua, K.B. & Kasri, A.R. 2011. Hand foot and mouth disease due to Enterovirus 71 in Malaysia. Virologica Sinica, 26(4): 221–228. DOI: https://doi.org/10.1007/s12250-011-3195-8
Coates, S.J., Davis, M.D.P. & Andersen, L.K. 2019. Temperature and humidity affect the incidence of hand, foot, and mouth disease: A systematic review of the literature - a report from the International Society of Dermatology Climate Change Committee. International Journal of Dermatology, 58(4): 388–399. DOI: https://doi.org/10.1111/ijd.14188
Criscuolo, E., Clementi, N., Mancini, N., Burioni, R., Miduri, M., Castelli, M. & Clementi, M. 2018. Synergy evaluation of anti-Herpes Simplex Virus type 1 and 2 compounds acting on different steps of virus life cycle. Antiviral Research, 151(3): 71–77. DOI: https://doi.org/10.1016/j.antiviral.2018.01.009
Cui, S., Wang, J., Fan, T., Qin, B., Guo, L., Lei, X., Wang, J., Wang, M. & Jin, Q. 2011. Crystal structure of human enterovirus 71 3C protease. Journal of Molecular Biology, 408(3): 449–461. DOI: https://doi.org/10.1016/j.jmb.2011.03.007
De Araújo, H.W.C., Fukushima, K. & Takaki, G.M.C. 2010. Prodigiosin production by Serratia marcescens UCP 1549 using renewable-resources as a low cost substrate. Molecules, 15(10): 6931–6940. DOI: https://doi.org/10.3390/molecules15106931
Du, X., Li, Y., Xia, Y.L., Ai, S. M., Liang, J., Sang, P., Ji, X.L. & Liu, S.Q. 2016. Insights into protein–ligand interactions: mechanisms, models, and methods. International Journal of Molecular Sciences, 17(2): 144. DOI: https://doi.org/10.3390/ijms17020144
Elahian, F., Moghimi, B., Dinmohammadi, F., Ghamghami, M., Hamidi, M. & Mirzaei, S.A. 2013. The anticancer agent prodigiosin is not a multidrug resistance protein substrate. DNA and Cell Biology, 32(3): 90–97. DOI: https://doi.org/10.1089/dna.2012.1902
Falah, N., Montserret, R., Lelogeais, V., Schuffenecker, I., Lina, B., Cortay, J.C. & Violot, S. 2012. Blocking human enterovirus 71 replication by targeting viral 2A protease. Journal of Antimicrobial Chemotherapy, 67(12): 2865–2869. DOI: https://doi.org/10.1093/jac/dks304
Fang, C.Y. & Liu, C.C. 2018. Recent development of Enterovirus A vaccine candidates for the prevention of hand, foot, and mouth disease. Expert Review of Vaccines, 17(9): 819–831. DOI: https://doi.org/10.1080/14760584.2018.1510326
Fong, S.Y., Mori, D., Rundi, C., Yap, J.F., Jikal, M., Latip, A.L.L.B.A., Johnny, V. & Ahmed, K. 2021. A five-year retrospective study on the epidemiology of hand, foot and mouth disease in Sabah, Malaysia. Scientific Reports, 11(1): 17814. DOI: https://doi.org/10.1038/s41598-021-96083-3
Guan, H., Tian, J., Qin, B., Wojdyla, J.A., Wang, B., Zhao, Z., Wang, M. & Cui, S. 2017. Crystal structure of 2C helicase from enterovirus 71. Science Advances, 3(4): e1602573. DOI: https://doi.org/10.1126/sciadv.1602573
Gupta, S.K., Singh, S., Nischal, A., Pant, K.K. & Seth, P.K. 2013. Molecular docking and simulation studies towards exploring antiviral compounds against envelope protein of Japanese encephalitis virus. Network Modeling and Analysis in Health Informatics and Bioinformatics, 2(4): 231–243. DOI: https://doi.org/10.1007/s13721-013-0040-z
Hayashi, K., Lee, J. B., Atsumi, K., Kanazashi, M., Shibayama, T., Okamoto, K., Kawahara, T. & Hayashi, T. 2019. In vitro and in vivo anti-herpes simplex virus activity of monogalactosyl diacylglyceride from Coccomyxa sp. KJ (IPOD FERM BP-22254), a green microalga. PLoS ONE, 14(7): e0219305. DOI: https://doi.org/10.1371/journal.pone.0219305
Hubbard, R. & Rimington, C. 1950. The biosynthesis of prodigiosin, the tripyrrylmethene pigment from Bacillus prodigiosus (Serratia marcescens). Biochemical Journal, 46(2): 220–225. DOI: https://doi.org/10.1042/bj0460220
Kim, B., Moon, S., Bae, G.R., Lee, H., Pai, H. & Oh, S.H. 2018. Factors associated with severe neurologic complications in patients with either hand-foot-mouth disease or herpangina: A nationwide observational study in South Korea, 2009-2014. PLOS ONE, 13(8): e0201726. DOI: https://doi.org/10.1371/journal.pone.0201726
Kuo, C.J., Shie, J.J., Fang, J.M., Yen, G.R., Hsu, J.T.A., Liu, H.G., Tseng, S.N., Chang, S.C., Lee, C.Y., Shih, S.R. & Liang, P.H. 2008. Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents. Bioorganic and Medicinal Chemistry, 16(15): 7388–7398. DOI: https://doi.org/10.1016/j.bmc.2008.06.015
Lapenda, J.C., Silva, P.A., Vicalvi, M.C., Sena, K.X.F.R. & Nascimento, S.C. 2015. Antimicrobial activity of prodigiosin isolated from Serratia marcescens UFPEDA 398. World Journal of Microbiology and Biotechnology, 31(2): 399–406. DOI: https://doi.org/10.1007/s11274-014-1793-y
Lapenda, L.J., Maciel, C., Xavier, H., Alves, da S.C. & Takaki, G.M.C. 2014. Production and toxicological evaluation of prodigiosin from Serratia marcescens UCP/WFCC1549 on mannitol solid medium. International Journal of Applied Research in Natural Products, 7(2): 32–38.
Lehár, J., Krueger, A.S., Avery, W., Heilbut, A.M., Johansen, L.M., Price, E.R., Rickles, R.J., Short Iii, G.F., Staunton, J.E., Jin, X. & Lee, M.S. 2009. Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nature Biotechnology, 27(7): 659–666. DOI: https://doi.org/10.1038/nbt.1549
Lei, X., Cui, S., Zhao, Z. & Wang, J. 2015. Etiology, pathogenesis, antivirals and vaccines of hand, foot, and mouth disease. National Science Review, 2(3): 268–284. DOI: https://doi.org/10.1093/nsr/nwv038
Li, M.L., Lin, J.Y., Chen, B.S., Weng, K.F., Shih, S.R., Calderon, J.D., Tolbert, B.S. & Brewer, G. 2019. EV71 3C protease induces apoptosis by cleavage of hnRNP A1 to promote apaf-1 translation. PLOS ONE, 14(9): e0221048. DOI: https://doi.org/10.1371/journal.pone.0221048
Li, M. L., Shih, S. R., Tolbert, B. S. & Brewer, G. 2021. Enterovirus a71 vaccines. Vaccines, 9(3): 1–10. DOI: https://doi.org/10.3390/vaccines9030199
Li, Y., Yu, J., Qi, X. & Yan, H. 2019. Monoclonal antibody against EV71 3Dpol inhibits the polymerase activity of RdRp and virus replication. BMC Immunology, 20(1): 6. DOI: https://doi.org/10.1186/s12865-019-0288-x
Li, Z., Cui, B., Liu, X., Wang, L., Xian, Q., Lu, Z., Liu, S., Cao, Y. & Zhao, Y. 2020. Virucidal activity and the antiviral mechanism of acidic polysaccharides against Enterovirus 71 infection in vitro. Microbiology and Immunology, 64(3): 189–201. DOI: https://doi.org/10.1111/1348-0421.12763
Lin, B., He, S., Yim, H.J., Liang, T.J. & Hu, Z. 2016. Evaluation of antiviral drug synergy in an infectious HCV system. Antiviral Therapy, 21(7): 595–603. DOI: https://doi.org/10.3851/IMP3044
Lin, C., Jia, X., Fang, Y., Chen, L., Zhang, H., Lin, R. & Chen, J. 2019. Enhanced production of prodigiosin by Serratia marcescens FZSF02 in the form of pigment pellets. Electronic Journal of Biotechnology, 40: 58–64. DOI: https://doi.org/10.1016/j.ejbt.2019.04.007
Lin, D., Jiang, S., Zhang, A., Wu, T., Qian, Y. & Shao, Q. 2022. Structural derivatization strategies of natural phenols by semi-synthesis and total-synthesis. Natural Products and Bioprospecting, 12(1): 1–29. DOI: https://doi.org/10.1007/s13659-022-00331-6
Lin, J.Y., Kung, Y.A. & Shih, S.R. 2019. Antivirals and vaccines for Enterovirus A71. Journal of Biomedical Science, 26(1): 1–10. DOI: https://doi.org/10.1186/s12929-019-0560-7
Littler, D.R., Liu, M., McAuley, J.L., Lowery, S.A., Illing, P.T., Gully, B.S., Purcell, A.W., Chandrashekaran, I.R., Perlman, S., Purcell, D.F. & Quinn, R.J. 2021. A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein. Journal of Biological Chemistry, 297(6): 101362. DOI: https://doi.org/10.1016/j.jbc.2021.101362
Mashiach, E., Schneidman-Duhovny, D., Andrusier, N., Nussinov, R. & Wolfson, H.J. 2008. FireDock: a web server for fast interaction refinement in molecular docking. Nucleic acids research, 36(Web Server issue): 229–232. DOI: https://doi.org/10.1093/nar/gkn186
May, A. & Zacharias, M. 2005. Accounting for global protein deformability during protein-protein and protein-ligand docking. Biochimica et Biophysica Acta - Proteins and Proteomics, 1754(1–2): 225–231. DOI: https://doi.org/10.1016/j.bbapap.2005.07.045
Mu, Z., Wang, B., Zhang, X., Gao, X., Qin, B., Zhao, Z. & Cui, S. 2013. Crystal structure of 2A proteinase from hand, foot and mouth disease virus. Journal of Molecular Biology, 425(22): 4530–4543. DOI: https://doi.org/10.1016/j.jmb.2013.08.016
Nakashima, T., Kurachi, M., Kato, Y., Yamaguchi, K. & Oda, T. 2005. Characterization of bacterium isolated from the sediment at coastal area of Omura Bay in Japan and several biological activities of pigment produced by this isolate. Microbiology and Immunology, 49(5): 407–415. DOI: https://doi.org/10.1111/j.1348-0421.2005.tb03744.x
Peele, K.A., Potla Durthi, C., Srihansa, T., Krupanidhi, S., Ayyagari, V.S., Babu, D.J., Indira, M., Reddy, A.R. & Venkateswarulu, T.C. 2020. Molecular docking and dynamic simulations for antiviral compounds against SARS-CoV-2: A computational study. Informatics in Medicine Unlocked, 19: 100345. DOI: https://doi.org/10.1016/j.imu.2020.100345
Pourianfar, H.R. & Grollo, L. 2015. Development of antiviral agents toward enterovirus 71 infection. Journal of Microbiology, Immunology and Infection, 48(1): 1–8. DOI: https://doi.org/10.1016/j.jmii.2013.11.011
Rabenau, H.F., Schwebke, I., Blümel, J., Eggers, M., Glebe, D., Rapp, I., Sauerbrei, A., Steinmann, E., Steinmann, J., Willkommen, H. & Wutzler, P . 2020. Guideline for testing chemical disinfectants regarding their virucidal activity within the field of human medicine. Bundesgesundheitsblatt, 63(5): 645–655. DOI: https://doi.org/10.1007/s00103-020-03115-w
Rasti, M., Khanbabaei, H. & Teimoori, A. 2019. An update on Enterovirus 71 infection and interferon type I response. Reviews in Medical Virology, 29(1): 2016. DOI: https://doi.org/10.1002/rmv.2016
Ren, J., Wang, X., Zhu, L., Hu, Z., Gao, Q., Yang, P., Li, X., Wang, J., Shen, X., Fry, E.E. & Rao, Z. 2015. Structures of Coxsackievirus A16 capsids with native antigenicity: Implications for particle expansion, receptor binding, and immunogenicity. Journal of Virology, 89(20): 10500–10511. DOI: https://doi.org/10.1128/JVI.01102-15
Schneidman-Duhovny, D., Inbar, Y., Nussinov, R. & Wolfson, H.J. 2005. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Research, 33(2): 363–367. DOI: https://doi.org/10.1093/nar/gki481
Shang, L., Xu, M. & Yin, Z. 2013. Antiviral drug discovery for the treatment of enterovirus 71 infections. Antiviral Research, 97(2): 183–194. DOI: https://doi.org/10.1016/j.antiviral.2012.12.005
Shih, S.R., Tsai, K.N., Li, Y.S., Chueh, C.C. & Chan, E.C. 2003. Inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis. Journal of Medical Virology, 70(1): 119–125. DOI: https://doi.org/10.1002/jmv.10363
Song, M.J., Bae, J., Lee, D.S., Kim, C.H., Kim, J.S., Kim, S.W. & Hong, S.I. 2006. Purification and characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. Journal of Bioscience and Bioengineering, 101(2): 157–161. DOI: https://doi.org/10.1263/jbb.101.157
Su, X., Wang, Q., Wen, Y., Jiang, S. & Lu, L. 2020. Protein- and peptide-based virus inactivators: Inactivating viruses before their entry into cells. Frontiers in Microbiology, 11: 1063. DOI: https://doi.org/10.3389/fmicb.2020.01063
Suba, K., Stalin, A., Girija, A. & Raguraman, R. 2013. Homology modeling and docking analysis of prodigiosin from Serratia marcescens. Elixir International Journal, 55: 12897–12902.
Sun, B.J., Chen, H.J., Chen, Y., An, X.D. & Zhou, B.S. 2018. The risk factors of acquiring severe hand, Foot, and mouth disease: A meta-analysis. Canadian Journal of Infectious Diseases and Medical Microbiology, 2018 (eCollection): 1–12. DOI: https://doi.org/10.1155/2018/2751457
Tan, C.W., Poh, C.L., Sam, I.-C. & Chan, Y.F. 2013. Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor. Journal of Virology, 87(1): 611–620. DOI: https://doi.org/10.1128/JVI.02226-12
Tan, Y.W., Ang, M.J.Y., Lau, Q.Y., Poulsen, A., Ng, F.M., Then, S.W., Peng, J., Hill, J., Hong, W.J., Chia, C.S.B. & Chu, J.J.H. 2016. Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease. Scientific Reports, 6(9): 1–9. DOI: https://doi.org/10.1038/srep33663
van der Linden, L., Wolthers, K.C. & van Kuppeveld, F.J.M. 2015. Replication and inhibitors of enteroviruses and parechoviruses. Viruses, 7(8): 4529–4562. DOI: https://doi.org/10.3390/v7082832
Wang, T., Wang, B., Huang, H., Zhang, C., Zhu, Y., Pei, B., Cheng, C., Sun, L., Wang, J., Jin, Q. & Zhao, Z. 2017. Enterovirus 71 protease 2Apro and 3Cpro differentially inhibit the cellular endoplasmic reticulum-associated degradation (ERAD) pathway via distinct mechanisms, and enterovirus 71 hijacks ERAD component p97 to promote its replication. PLoS Pathogens, 13(10): e1006674. DOI: https://doi.org/10.1371/journal.ppat.1006674
Wei, Y.H. & Chen, W.C. 2005. Enhanced production of prodigiosin-like pigment from Serratia marcescens SMAR by medium improvement and oil-supplementation strategies. Journal of Bioscience and Bioengineering, 99(6): 616–622. DOI: https://doi.org/10.1263/jbb.99.616
Wen, W., Qi, Z. & Wang, J. 2020. The function and mechanism of Enterovirus 71 (EV71) 3C protease. Current Microbiology, 77(9): 1968–1975. DOI: https://doi.org/10.1007/s00284-020-02082-4
Wlodawer, A., Minor, W., Dauter, Z. & Jaskolski, M. 2008. Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures. FEBS Journal, 275(1): 1–21. DOI: https://doi.org/10.1111/j.1742-4658.2007.06178.x
World Health Organization. 2018. Hand, foot and mouth disease situation update 2018 [WWW Document]. URL https://apps.who.int/iris/handle/10665/274107 (accessed 9.12.2022).
Xia, H., Wang, P., Wang, G.C., Yang, J., Sun, X., Wu, W., Qiu, Y., Shu, T., Zhao, X., Yin, L. & Qin, C. F. 2015. Human enterovirus nonstructural protein 2CATPase functions as both an RNA helicase and ATP-independent RNA chaperone. PLOS Pathogens, 11(7): e1005067. DOI: https://doi.org/10.1371/journal.ppat.1005067
Xu, W.F., Wu, N.N., Wu, Y.W., Qi, Y.X., Wei, M.Y., Pineda, L.M., Ng, M.G., Spadafora, C., Zheng, J.Y., Lu, L. & Wang, C.Y. 2022. Structure modification, antialgal, antiplasmodial, and toxic evaluations of a series of new marine-derived 14-membered resorcylic acid lactone derivatives. Marine Life Science and Technology, 4(1): 88–97. DOI: https://doi.org/10.1007/s42995-021-00103-0
Yang, Y., Xiu, J., Zhang, X., Zhang, L., Yan, K., Qin, C. & Liu, J. 2012. Antiviral effect of matrine against human enterovirus 71. Molecules, 17(9): 10370–10376. DOI: https://doi.org/10.3390/molecules170910370
Yuan, J., Shen, L., Wu, J., Zou, X., Gu, J., Chen, J. & Mao, L. 2018. Enterovirus A71 proteins: Structure and function. Frontiers in Microbiology, 9(286): 1–8. DOI: https://doi.org/10.3389/fmicb.2018.00286
Yuan, S., Li, G., Wang, Y., Gao, Q., Wang, Y., Cui, R., Altmeyer, R. & Zou, G. 2016. Identification of positively charged residues in Enterovirus 71 capsid protein VP1 essential for production of infectious particles. Journal of Virology, 90(2): 741–752. DOI: https://doi.org/10.1128/JVI.02482-15
Zhang, C., Zhai, S., Li, X., Zhang, Q., Wu, L., Liu, Y., Jiang, C., Zhou, H., Li, F., Zhang, S. & Su, G. 2014. Synergistic action by multi-targeting compounds produces a potent compound combination for human NSCLC both in vitro and in vivo. Cell Death and Disease, 5(3): e1138–e1138. DOI: https://doi.org/10.1038/cddis.2014.76
Zhu, Q.C., Wang, Y., Liu, Y.P., Zhang, R.Q., Li, X., Su, W.H., Long, F., Luo, X.D. & Peng, T. 2011. Inhibition of enterovirus 71 replication by chrysosplenetin and penduletin. European Journal of Pharmaceutical Sciences, 44(3): 392–398. DOI: https://doi.org/10.1016/j.ejps.2011.08.030
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