Skip to main content

Advertisement

Springer Nature Link
Log in

Potent SARS-CoV-2 3C-like protease inhibitor ( +)-eupenoxide-3,6-diketone (IC50: 0.048 μM) was synthesized based on ( +)-eupenoxide; lead from ( +)-eupenoxide analogs study by endophytic fermentation

  • Original Paper
  • Published:
Journal of Natural Medicines Aims and scope Submit manuscript

Abstract

Since the coronavirus disease 2019 (COVID-19) outbreak, research has been conducted on treatment and countermeasures against the causative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the development of new seeds is urgently needed because viruses have the characteristic of becoming resistant through mutation. We hypothesize that endophytes produce antiviral substances to combat foreign viruses in host plants. According to this hypothesis, the seeds of therapeutic agents for infectious diseases could be obtained from endophytes by culture experiments. This report found that Aspergillus sp. endophyte isolated from Catharanthus roseus produced ( +)-eupenoxide and its 3-ketone form with anti-SARS-CoV-2 activity. In addition, ( +)-eupenoxide-3,6-diketon was discovered as a new compound with potent 3C-like protease inhibitory activity (IC50: 0.048 μM) by synthesis based on ( +)-eupenoxide. This finding could be an important evidence that endophytic fungi symbiosis with medicinal plants is useful as antiviral producers.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Wani ZA, Ashraf N, Mohiuddin T, Riyaz-Ul-Hassan S (2015) Plant-endophyte symbiosis, an ecological perspective. Appl Microbiol Biotechnol 99:2955–2965. https://doi.org/10.1007/S00253-015-6487-3/FIGURES/3

    Article  PubMed  CAS  Google Scholar 

  2. Saikkonen K, Wäli P, Helander M, Faeth SH (2004) Evolution of endophyte–plant symbioses. Trends Plant Sci 9:275–280. https://doi.org/10.1016/J.TPLANTS.2004.04.005

    Article  PubMed  CAS  Google Scholar 

  3. Arachevaleta M, Bacon CW, Hoveland CS, Radcliffe DE (1989) Effect of the tall fescue endophyte on plant response to environmental stress. Agron J 81:83–90. https://doi.org/10.2134/agronj1989.00021962008100010015x

    Article  Google Scholar 

  4. Nejidat A (1987) Effect of ophiobolin A on stomatal movement: role of calmodulin. Plant Cell Physiol 28:455–460

    CAS  Google Scholar 

  5. Kanda K, Hirai Y, Koga H, Hasegawa K (1994) Endophyte-enhanced resistance in perennial ryegrass and tall fescue to bluegrass webworm, parapediasia teterrella. Japanese J Appl Entomol Zool 38:141–145. https://doi.org/10.1303/jjaez.38.141

    Article  Google Scholar 

  6. Trivedi P, Leach JE, Tringe SG et al (2020) Plant–microbiome interactions: from community assembly to plant health. Nat Rev Microbiol 1811(18):607–621. https://doi.org/10.1038/s41579-020-0412-1

    Article  CAS  Google Scholar 

  7. Maehara S, Nakajima S, Watashi K et al (2023) Anti-SARS-CoV-2 agents in artemisia endophytic fungi and their abundance in artemisia vulgaris tissue. J Fungi 9:905. https://doi.org/10.3390/jof9090905

    Article  CAS  Google Scholar 

  8. Kumar A, Patil D, Rajamohanan PR, Ahmad A (2013) Isolation, purification and characterization of vinblastine and vincristine from endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus. PLoS One. https://doi.org/10.1371/JOURNAL.PONE.0071805

    Article  PubMed  PubMed Central  Google Scholar 

  9. Duke RK, Rickards RW (1984) Stereospecific total synthesis of the cyclohexene oxide antibiotic eupenoxide. J Org Chem 49:1898–1904. https://doi.org/10.1021/JO00185A010/ASSET/JO00185A010.FP.PNG_V03

    Article  CAS  Google Scholar 

  10. Mehta G, Roy S, Davis RA (2008) On the stereostructures of (+)-eupenoxide and (−)-3′,4′-dihydrophomoxide: a caveat on the spectral comparisons of oxygenated cyclohexenoids. Tetrahedron Lett 49:5162–5164. https://doi.org/10.1016/J.TETLET.2008.06.076

    Article  CAS  Google Scholar 

  11. Myobatake Y, Takemoto K, Kamisuki S et al (2014) Cytotoxic alkylated hydroquinone, phenol, and cyclohexenone derivatives from aspergillus violaceofuscus Gasperini. J Nat Prod 77:1236–1240. https://doi.org/10.1021/NP401017G/SUPPL_FILE/NP401017G_SI_001.PDF

    Article  PubMed  CAS  Google Scholar 

  12. Li C, Porco JA (2005) Synthesis of epoxyquinol A and related molecules: probing chemical reactivity of epoxyquinol dimers and 2H-pyran precursors. J Org Chem 70:6053–6065. https://doi.org/10.1021/JO050897O

    Article  PubMed  CAS  Google Scholar 

  13. Mehta G, Roy S (2004) Enantioselective total synthesis of (+)-eupenoxide and (+)-phomoxide: revision of structures and assignment of absolute configuration. Org Lett 6:2389–2392. https://doi.org/10.1021/OL0492288/SUPPL_FILE/OL0492288SI20040604_101136.PDF

    Article  PubMed  CAS  Google Scholar 

  14. Kleinke AS, Li C, Rabasso N, Porco JA (2006) Total synthesis of the interleukin-1β converting enzyme inhibitor EI-1941-2 using tandem oxa-electrocyclization/oxidation. Org Lett 8:2847–2850. https://doi.org/10.1021/ol060954f

    Article  PubMed  CAS  Google Scholar 

  15. Kakeya H, Miyake Y, Shoji M et al (2003) Novel non-peptide inhibitors targeting death receptor-Mediated apoptosis. Bioorg Med Chem Lett 13:3743–3746. https://doi.org/10.1016/J.BMCL.2003.08.003

    Article  PubMed  CAS  Google Scholar 

  16. Fujita K, Ishikawa F, Kakeya H (2014) Biosynthetic origins of the epoxyquinone skeleton in epoxyquinols A and B. J Nat Prod 77:2707–2710. https://doi.org/10.1021/NP5004615/SUPPL_FILE/NP5004615_SI_001.PDF

    Article  PubMed  Google Scholar 

  17. Li P, Takei R, Takahashi K, Nabeta K (2007) Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae. Phytochemistry 68:819–823. https://doi.org/10.1016/J.PHYTOCHEM.2006.12.006

    Article  PubMed  CAS  Google Scholar 

  18. Tsukamoto S, Hirota H, Imachi M et al (2005) Himeic acid A: a new ubiquitin-activating enzyme inhibitor isolated from a marine-derived fungus, Aspergillus sp. Bioorg Med Chem Lett 15:191–194. https://doi.org/10.1016/J.BMCL.2004.10.012

    Article  PubMed  CAS  Google Scholar 

  19. Sakekar AA, Gaikwad SR, Punekar NS (2021) Protein expression and secretion by filamentous fungi. J Biosci 461(46):1–18. https://doi.org/10.1007/S12038-020-00120-8

    Article  Google Scholar 

  20. Uzaki M, Mori T, Sato M et al (2024) Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus. New Phytol 242:1156–1171. https://doi.org/10.1111/NPH.19662

    Article  PubMed  CAS  Google Scholar 

  21. Davis RA, Andjic V, Kotiw M, Shivas RG (2005) Phomoxins B and C: polyketides from an endophytic fungus of the genus Eupenicillium. Phytochemistry 66:2771–2775. https://doi.org/10.1016/J.PHYTOCHEM.2005.09.004

    Article  PubMed  CAS  Google Scholar 

  22. Dramae A, Bunbamrung N, Intaraudom C et al (2024) Polyoxygenated cyclohexenoids from the endophytic fungus, Aspergillus aculeatus BCC69431 and biological properties. Tetrahedron 150:133753. https://doi.org/10.1016/J.TET.2023.133753

    Article  CAS  Google Scholar 

  23. Ohashi H, Watashi K, Saso W et al (2021) Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment. iScience. https://doi.org/10.1016/j.isci.2021.102367

    Article  PubMed  PubMed Central  Google Scholar 

  24. Liu H, Iketani S, Zask A et al (2022) Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19. Nat Commun 131(13):1–16. https://doi.org/10.1038/s41467-022-29413-2

    Article  CAS  Google Scholar 

  25. Ramos-Guzmán CA, Ruiz-Pernía JJ, Tuñón I (2021) Inhibition mechanism of SARS-CoV-2 main protease with ketone-based inhibitors unveiled by multiscale simulations: insights for improved designs**. Angew Chemie Int Ed 60:25933–25941. https://doi.org/10.1002/ANIE.202110027

    Article  Google Scholar 

  26. Unoh Y, Uehara S, Nakahara K et al (2022) Discovery of S-217622, a noncovalent oral SARS-CoV-2 3CL protease inhibitor clinical candidate for treating COVID-19. J Med Chem 65:6499–6512. https://doi.org/10.1021/ACS.JMEDCHEM.2C00117

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Bai B, Arutyunova E, Khan MB et al (2021) Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors. RSC Med Chem 12:1722–1730. https://doi.org/10.1039/D1MD00247C

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Gao S, Sylvester K, Song L et al (2022) Discovery and crystallographic studies of trisubstituted piperazine derivatives as non-covalent SARS-CoV-2 main protease inhibitors with high target specificity and low toxicity. J Med Chem 65:13343–13364. https://doi.org/10.1021/ACS.JMEDCHEM.2C01146/SUPPL_FILE/JM2C01146_SI_002.CSV

    Article  PubMed  CAS  Google Scholar 

  29. Liu H, Ye F, Sun Q et al (2020) Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro. bioRxiv. https://doi.org/10.1101/2020.04.10.035824

    Article  PubMed  PubMed Central  Google Scholar 

  30. Shi TH, Huang YL, Chen CC et al (2020) Andrographolide and its fluorescent derivative inhibit the main proteases of 2019-nCoV and SARS-CoV through covalent linkage. Biochem Biophys Res Commun 533:467–473. https://doi.org/10.1016/J.BBRC.2020.08.086

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Maehara S, Fathoni A, Tagawa M et al (2023) Environmental differences between Japan and Indonesia provide endophyte diversity associated with Artemisia plant and variety of artemisinin derivatives in microbial conversion. J Nat Med 77:916–927. https://doi.org/10.1007/s11418-023-01709-7

    Article  PubMed  CAS  Google Scholar 

  32. Pracht P, Grimme S, Bannwarth C et al (2024) CREST-a program for the exploration of low-energy molecular chemical space. J Chem Phys. https://doi.org/10.1063/5.0197592

    Article  PubMed  Google Scholar 

  33. Bannwarth C, Ehlert S, Grimme S (2019) GFN2-xTB-an accurate and broadly parametrized self-consistent tight-binding quantum chemical method with multipole electrostatics and density-dependent dispersion contributions. J Chem Theory Comput 15:1652–1671. https://doi.org/10.1021/ACS.JCTC.8B01176

    Article  PubMed  CAS  Google Scholar 

  34. Grimme S, Bohle F, Hansen A et al (2021) Efficient quantum chemical calculation of structure ensembles and free energies for nonrigid molecules. J Phys Chem A 125:4039–4054. https://doi.org/10.1021/ACS.JPCA.1C00971

    Article  PubMed  CAS  Google Scholar 

  35. Neese F (2012) The ORCA program system. Wiley Interdiscip Rev Comput Mol Sci 2:73–78. https://doi.org/10.1002/WCMS.81

    Article  CAS  Google Scholar 

  36. Neese F (2018) Software update: the ORCA program system, version 4.0. Wiley Interdiscip Rev Comput Mol Sci 8:e1327. https://doi.org/10.1002/WCMS.1327

    Article  Google Scholar 

  37. Neese F, Wennmohs F, Becker U, Riplinger C (2020) The ORCA quantum chemistry program package. J Chem Phys. https://doi.org/10.1063/5.0004608

    Article  PubMed  Google Scholar 

  38. Grimme S, Bannwarth C, Dohm S et al (2017) Fully automated quantum-chemistry-based computation of spin-spin-coupled nuclear magnetic resonance spectra. Angew Chem Int Ed Engl 56:14763–14769. https://doi.org/10.1002/ANIE.201708266

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Lu W, Zhang J, Huang W et al (2024) DynamicBind: predicting ligand-specific protein-ligand complex structure with a deep equivariant generative model. Nat Commun. https://doi.org/10.1038/S41467-024-45461-2

    Article  PubMed  PubMed Central  Google Scholar 

  40. Pettersen EF, Goddard TD, Huang CC et al (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. https://doi.org/10.1002/JCC.20084

    Article  PubMed  CAS  Google Scholar 

  41. Laskowski RA, Swindells MB (2011) LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 51:2778–2786. https://doi.org/10.1021/CI200227U/ASSET/IMAGES/MEDIUM/CI-2011-00227U_0006.GIF

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We appreciate the technical help in measuring QTOF-MS for Prof. Masaya Ohta (Fukuyama University, Hiroshima, Japan). In addition, we appreciate our collaborator Prof. Hiroshi Kamitakahara (Kyoto University, Kyoto, Japan), as a WP3 Leader of Japan-ASEAN Science, Technology and Innovation Platform (JASTIP).

Funding

This research was funded by the Japan Science and Technology Agency Strategic International Collaborative Research Program (SICORP, Japan), grant number JPMJSC15H1; JST MIRAI program, grant number JPMJMI22G1; JSPS KAKENHI, grant numbers 23K06189 and 24K02290; and the Japan Agency for Medicinal Research and Development (AMED), grant number JP23fk0108590.

Author information

Authors and Affiliations

  1. Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Sanzo, 1 Gakuen-cho, Fukuyama, Hiroshima, 729-0292, Japan

    Shoji Maehara, Moeka Kumamoto & Toshiyuki Hata

  2. Department of Virology II, National Institute of Infectious Diseases, Toyama 1‐23‐1, Shinjuku-ku, Tokyo, 162‐8640, Japan

    Shogo Nakajima & Koichi Watashi

  3. Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo, 162-8640, Japan

    Shogo Nakajima & Koichi Watashi

  4. Common Resources Center, Fukuyama University, 985-1, Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292, Japan

    Yuhzo Hieda

Authors
  1. Shoji Maehara
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Moeka Kumamoto
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Shogo Nakajima
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Yuhzo Hieda
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Koichi Watashi
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Toshiyuki Hata
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

All authors contributed to the study’s conception and design. Shoji Maehara and Moeka Kumamoto performed material preparation, data collection about compounds, and measurement and analysis of 3CL protease inhibition activity. Yuzoh Hieda synthesized ( +)-eupenoxide derivatives. Toshiyuki Hata performed computational analysis. Shogo Nakajima and Koichi Watashi evaluated the antiviral activity of SARS-CoV-2 using infected cells. Shoji Maehara wrote the first draft of the manuscript and all authors commented on previous versions of the manuscript.

Corresponding author

Correspondence to Shoji Maehara.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest associated with this manuscript.

Additional information

Publisher's Note

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

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 2634 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maehara, S., Kumamoto, M., Nakajima, S. et al. Potent SARS-CoV-2 3C-like protease inhibitor ( +)-eupenoxide-3,6-diketone (IC50: 0.048 μM) was synthesized based on ( +)-eupenoxide; lead from ( +)-eupenoxide analogs study by endophytic fermentation. J Nat Med 79, 357–370 (2025). https://doi.org/10.1007/s11418-024-01874-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11418-024-01874-3

Keywords