High-content screening of diterpenoids from Isodon species as autophagy modulators and the functional study of their antiviral activities

Abstract

Autophagy is a conserved lysosomal degradation process, and abnormal autophagy has been associated with various pathological processes, e.g., neurodegeneration, cancer, and pathogen infection. Small chemical modulators of autophagy show the potential to treat autophagy-associated diseases. Diterpenoids, nature products found in various plants, exhibit a wide range of bioactivity, and we have recently isolated and characterized over 150 diterpenoids from Isodon species distributed in China. Here, we applied a high-content fluorescence imaging-based assay to assess these diterpenoids’ ability to affect autophagic flux in HeLa cells. We found that enanderinanin J, an ent-kauranoid dimer, is an autophagy inhibitor, manifested by its ability to increase lysosomal pH and inhibit the fusion between autophagosomes and lysosomes. Autophagy has been shown to be either positively or negatively involved in the life cycle of Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and enterovirus-A71 (EV-A71). We found that enanderinanin J significantly inhibited the infection of ZIKV, DENV, JEV, or EV-A71. Interestingly, although ATG5 knockdown inhibited ZIKV or JEV infection, enanderinanin J further inhibited the infection of ZIKV or JEV in ATG5-knockdown cells. Taken together, our data indicate that enanderinanin J inhibits autophagosome-lysosome fusion and is a potential antiviral agent.

Graphical abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Data availability

Data and materials are available and transparent.

Abbreviations

ARJ:

acetonide of Rubescensin J

BAF:

bafilomycin A1

LC3:

microtubule-associated protein 1 light chain 3

GFP:

green fluorescence protein

RFP:

red fluorescence protein

JEV:

Japanese encephalitis virus

ZIKV:

Zika virus

EV-A17:

enterovirus A17

DENV:

Dengue virus

(+)ss RNA viruses:

positive-sense single-stranded RNA viruses

References

  1. Acharya B, Gyeltshen S, Chaijaroenkul W, Na-Bangchang K. Significance of autophagy in dengue virus infection: a brief review. Am J Trop Med Hyg. 2019;100:783–90.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Ahmad L, Mostowy S, Sancho-Shimizu V. Autophagy-virus interplay: from cell biology to human disease. Front Cell Dev Biol. 2018;6:155.

    PubMed  PubMed Central  Article  Google Scholar 

  3. Cao B, Parnell LA, Diamond MS, Mysorekar IU. Inhibition of autophagy limits vertical transmission of Zika virus in pregnant mice. J Exp Med. 2017;214:2303–13.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Daep CA, Munoz-Jordan JL, Eugenin EA. Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus. J Neuro-Oncol. 2014;20:539–60.

    CAS  Google Scholar 

  5. Dash S, Aydin Y, Wu T. Integrated stress response in hepatitis C promotes Nrf2-related chaperone-mediated autophagy: a novel mechanism for host-microbe survival and HCC development in liver cirrhosis[C]//Seminars in cell & developmental biology. Academic Press. 2020;101:20–35.

    CAS  Google Scholar 

  6. Davis-Kaplan SR, Ward DM, Shiflett SL, Kaplan J. Genome-wide analysis of iron-dependent growth reveals a novel yeast gene required for vacuolar acidification. J Biol Chem. 2004;279:4322–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. de Souza NJ, Dohadwalla AN, Reden J. Forskolin: a labdane diterpenoid with antihypertensive, positive inotropic, platelet aggregation inhibitory, and adenylate cyclase activating properties. Med Res Rev. 1983;3:201–19.

    PubMed  Article  PubMed Central  Google Scholar 

  8. Echavarria-Consuegra L, Smit JM, Reggiori F. Role of autophagy during the replication and pathogenesis of common mosquito-borne flavi- and alphaviruses. Open Biol. 2019;9:190009.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Eskelinen EL. Maturation of autophagic vacuoles in Mammalian cells. Autophagy. 2005;1:1–10.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. Fu Y, Xu W, Chen D, Feng C, Zhang L, Wang X, et al. Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication. Antivir Res. 2015;124:43–53.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. Fujita E, Node M. Diterpenoids of Rabdosia species[M]//Fortschritte der Chemie organischer Naturstoffe/Progress in the Chemistry of Organic Natural Products. Vienna: Springer; 1984. p. 77–157.

    Google Scholar 

  12. Gratton R, Agrelli A, Tricarico PM, et al. Autophagy in Zika virus infection: a possible therapeutic target to counteract viral replication[J]. International journal of molecular sciences. 2019;20(5):1048.

    CAS  PubMed Central  Article  Google Scholar 

  13. Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, et al. Biology of Zika virus infection in human skin cells. J Virol. 2015;89:8880–96.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Huang L, Yue J. The interplay of autophagy and enterovirus[C]//Seminars in Cell & Developmental Biology. Academic Press, 2019.

  15. Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol. 2011;8:528–39.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. Jin R, Zhu W, Cao S, Chen R, Jin H, Liu Y, et al. Japanese encephalitis virus activates autophagy as a viral immune evasion strategy. PLoS One. 2013;8:e52909.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Jones-Jamtgaard KN, Wozniak AL, Koga H, Ralston R, Weinman SA. Hepatitis C virus infection increases autophagosome stability by suppressing lysosomal fusion through an Arl8b-dependent mechanism. J Biol Chem. 2019;294:14257–66.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, et al. An autophagic flux probe that releases an internal control. Mol Cell. 2016;64:835–49.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. Kawai A, Uchiyama H, Takano S, Nakamura N, Ohkuma S. Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy. 2007;3:154–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. Ke PY. The multifaceted roles of autophagy in flavivirus-host interactions. Int J Mol Sci. 2018;19(12):3940.

    PubMed Central  Article  Google Scholar 

  21. Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer. 2005;5:886–97.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. Lai JKF, Sam IC, Verlhac P, Baguet J, Eskelinen EL, Faure M, et al. 2BC non-structural protein of enterovirus A71 interacts with SNARE proteins to trigger autolysosome formation. Viruses. 2017;9:169.

    PubMed Central  Article  CAS  Google Scholar 

  23. Lee YR, Lei HY, Liu MT, Wang JR, Chen SH, Jiang-Shieh YF, et al. Autophagic machinery activated by dengue virus enhances virus replication. Virology. 2008;374:240–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Lee YR, Wang PS, Wang JR, Liu HS. Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model. J Biomed Sci. 2014;21:80.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. Leung CH, Grill SP, Lam W, Han QB, Sun HD, Cheng YC. Novel mechanism of inhibition of NF-κB DNA-binding activity by diterpenoids isolated from Isodon rubescens. Mol Pharmacol. 2005;68:286–97.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. Li JK, Liang JJ, Liao CL, Lin YL. Autophagy is involved in the early step of Japanese encephalitis virus infection. Microbes Infect. 2012;14:159–68.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. Li C, Huang L, Sun W, Chen Y, He ML, Yue J, et al. Saikosaponin D suppresses enterovirus A71 infection by inhibiting autophagy. Signal Transduct Target Ther. 2019;4:4.

    PubMed  PubMed Central  Article  Google Scholar 

  28. Liang Q, Luo Z, Zeng J, Chen W, Foo SS, Lee SA, et al. Zika virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy. Cell Stem Cell. 2016;19:663–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Liu M, Wang WG, Sun HD, Pu JX. Diterpenoids from Isodon species: an update. Nat Prod Rep. 2017;34:1090–140.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. Liu Y, Gordesky-Gold B, Leney-Greene M, Weinbren NL, Tudor M, Cherry S. Inflammation-induced, STING-dependent autophagy restricts Zika virus infection in the Drosophila brain. Cell Host Microbe. 2018;24:57–68 e53.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Lu Y, Dong S, Hao B, Li C, Zhu K, Guo W, et al. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A. Autophagy. 2014;10:1895–905.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. Manders E, Verbeek F, Aten J. Measurement of co-localization of objects in dual-colour confocal images. J Microsc. 1993;169:375–82.

    Article  Google Scholar 

  33. Manfredi JJ, Horwitz SB. Taxol: an antimitotic agent with a new mechanism of action. Pharmacol Ther. 1984;25:83–125.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. Marwaha R, Sharma M. DQ-Red BSA trafficking assay in cultured cells to assess cargo delivery to lysosomes. Bio Protoc. 2017;7:e2571.

    PubMed  PubMed Central  Article  Google Scholar 

  35. Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev. 2010;90:1383–435.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery. Antivir Res. 2009;81:6–15.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. Sharma M, Bhattacharyya S, Nain M, Kaur M, Sood V, Gupta V, et al. Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes. Autophagy. 2014;10:1637–51.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. Sharma M, Bhattacharyya S, Sharma KB, Chauhan S, Asthana S, Abdin MZ, et al. Japanese encephalitis virus activates autophagy through XBP1 and ATF6 ER stress sensors in neuronal cells. J Gen Virol. 2017;98:1027–39.

    CAS  PubMed  Article  Google Scholar 

  39. Sharma M, Sharma KB, Chauhan S, Bhattacharyya S, Vrati S, Kalia M. Diphenyleneiodonium enhances oxidative stress and inhibits Japanese encephalitis virus induced autophagy and ER stress pathways. Biochem Biophys Res Commun. 2018;502:232–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. Shen Y, Liang WJ, Shi YN, Kennelly EJ, Zhao DK. Structural diversity, bioactivities, and biosynthesis of natural diterpenoid alkaloids. Nat Prod Rep. 2020;37:763–96.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. Solomon T, Mallewa M. Dengue and other emerging flaviviruses. J Inf Secur. 2001;42:104–15.

    CAS  Google Scholar 

  42. Song Z, Xu Y, Bao L, Zhang L, Yu P, Qu Y, et al. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses. 2019;11:59.

    CAS  PubMed Central  Article  Google Scholar 

  43. Su W, Huang S, Zhu H, Zhang B, Wu X. Interaction between PHB2 and Enterovirus A71 VP1 induces autophagy and affects EV-A71 infection. Viruses. 2020;12:414.

    CAS  PubMed Central  Article  Google Scholar 

  44. Sun HDX, Y L, Jiang B. Diterpenoids from Isodon Species; 2001, P 3.

  45. Sun H-D, Huang S-X, Han Q-B. Diterpenoids from Isodon species and their biological activities. Nat Prod Rep. 2006;23:673–98.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. Tasaki T, Nukuzuma S, Takegami T. Impaired Japanese encephalitis virus replication in p62/SQSTM1 deficient mouse embryonic fibroblasts. Microbiol Immunol. 2016;60:708–11.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. Tong J, Yan X, Yu L. The late stage of autophagy: cellular events and molecular regulation. Protein Cell. 2010;1:907–15.

    PubMed  PubMed Central  Article  Google Scholar 

  48. White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 2012;12:401–10.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Wong HH, Sanyal S. Manipulation of autophagy by (+) RNA viruses. Semin Cell Dev Biol. 2019;8(7):674.

    Google Scholar 

  50. Wong HH, Sanyal S. Manipulation of autophagy by (+) RNA viruses. Semin Cell Dev Biol. 2020;101:3–11.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. Wu WKK, Yue J. Autophagy in host-microbe interactions. Semin Cell Dev Biol. 2020;101:1–2.

    PubMed  Article  PubMed Central  Google Scholar 

  52. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23:33–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010;12:814–22.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Yao R, Chen ZL, Zhou CC, Luo M, Shi XJ, Li JG, et al. Xerophilusin B induces cell cycle arrest and apoptosis in esophageal squamous cell carcinoma cells and does not cause toxicity in nude mice. J Nat Prod. 2015;78:10–6.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. Zhang H, Baehrecke EH. Eaten alive: novel insights into autophagy from multicellular model systems. Trends Cell Biol. 2015;25:376–87.

    PubMed  PubMed Central  Article  Google Scholar 

  56. Zhang S, Yi C, Li C, Zhang F, Peng J, Wang Q, et al. Chloroquine inhibits endosomal viral RNA release and autophagy-dependent viral replication and effectively prevents maternal to fetal transmission of Zika virus. Antivir Res. 2019;169:104547.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by the CAS-Croucher Funding Scheme (JY), Hong Kong Research Grant Council (RGC) grants (11101717 and 11103620 to JY), NSFC (21778045 and 32070702 to JY and 81673329 to TP), Shenzhen government research grant (JCYJ20160229165235739 and JCYJ20170413141331470 to JY), Sichuan Science and Technology Program (# 2019YJ063 to JY), and the Second Tibetan Plateau Scientific Expedition and Research (STEP) program (2019QZKK0502 to TP).

Author information

Affiliations

Authors

Contributions

L.H., Q.F., J-M.D., D.Y., and D.W. performed the experiments; T.P. and J.Y. wrote the manuscript; and J.Y. conceived the study and designed the experiments.

Corresponding authors

Correspondence to Pema-Tenzin Puno or Jianbo Yue.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

ESM 1

(DOCX 1703 kb)

ESM 2

(PDF 292 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, L., Fu, Q., Dai, JM. et al. High-content screening of diterpenoids from Isodon species as autophagy modulators and the functional study of their antiviral activities. Cell Biol Toxicol (2021). https://doi.org/10.1007/s10565-021-09580-6

Download citation

Keywords

  • Diterpenoids
  • Autophagy
  • Lysosome
  • Autophagosome
  • Enanderinanin J
  • Dengue virus (DENV)
  • Zika virus (ZIKV)
  • Japanese encephalitis virus (JEV)
  • Enterovirus-A71 (EV-A71)