Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Toremifene interacts with and destabilizes the Ebola virus glycoprotein

Nature volume 535, pages 169–172 (2016)Cite this article

Subjects

Abstract

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion1,2,3,4,5,6,7. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered8,9,10. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Summary of thermal-shift assays.
Figure 2: Overall structure.
Figure 3: Structure comparisons.
Figure 4: Inhibitor-binding site.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors have been deposited with the RCSB Protein Data Bank under accession codes 5JQ3 (native GP), 5JQ7 (GP-toremifene) and 5JQB (GP-ibuprofen).

References

  1. Takada, A. et al. A system for functional analysis of Ebola virus glycoprotein. Proc. Natl Acad. Sci. USA 94, 14764–14769 (1997)

    Article  ADS  CAS  Google Scholar 

  2. Hacke, M. et al. Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol. Nat. Commun. 6, 7688 (2015)

    Article  ADS  Google Scholar 

  3. Aleksandrowicz, P. et al. Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis. J. Infect. Dis. 204 (suppl. 3), S957–S967 (2011)

    Article  CAS  Google Scholar 

  4. Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343 (2011)

    Article  ADS  CAS  Google Scholar 

  5. Côté, M. et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection. Nature 477, 344–348 (2011)

    Article  ADS  Google Scholar 

  6. Nanbo, A. et al. Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog. 6, e1001121 (2010)

    Article  Google Scholar 

  7. Saeed, M. F., Kolokoltsov, A. A., Albrecht, T. & Davey, R. A. Cellular entry of Ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog. 6, e1001110 (2010)

    Article  Google Scholar 

  8. Lee, J. E. et al. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454, 177–182 (2008)

    Article  ADS  CAS  Google Scholar 

  9. Wang, H. et al. Ebola viral glycoprotein bound to its endosomal receptor Niemann-Pick C1. Cell 164, 258–268 (2016)

    Article  CAS  Google Scholar 

  10. Dias, J. M. et al. A shared structural solution for neutralizing ebolaviruses. Nat. Struct. Mol. Biol. 18, 1424–1427 (2011)

    Article  CAS  Google Scholar 

  11. World Health Organization. Ebola Situation Reports ; http://www.who.int/csr/disease/ebola/situation-reports/en/ (2016)

  12. Veljkovic, V. et al. In silico analysis suggests repurposing of ibuprofen for prevention and treatment of EBOLA virus disease. F1000Res . 4, 104 (2015)

    PubMed  Google Scholar 

  13. Edwards, M. R. et al. High-throughput minigenome system for identifying small-molecule inhibitors of Ebola virus replication. ACS Infect. Dis. 1, 380–387 (2015)

    Article  CAS  Google Scholar 

  14. Johansen, L. M. et al. FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Sci. Transl. Med. 5, 190ra79 (2013)

    Article  Google Scholar 

  15. Johansen, L. M. et al. A screen of approved drugs and molecular probes identifies therapeutics with anti-Ebola virus activity. Sci. Transl. Med. 7, 290ra89 (2015)

    Article  Google Scholar 

  16. Kouznetsova, J. et al. Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerg. Microbes Infect. 3, e84 (2014)

    CAS  Google Scholar 

  17. Lea, W. A. & Simeonov, A. Differential scanning fluorometry signatures as indicators of enzyme inhibitor mode of action: case study of glutathione S-transferase. PLoS One 7, e36219 (2012)

    Article  ADS  CAS  Google Scholar 

  18. Cimmperman, P. et al. A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophys. J. 95, 3222–3231 (2008)

    Article  ADS  CAS  Google Scholar 

  19. Cheng, H. et al. Inhibition of Ebola and Marburg virus entry by G protein-coupled receptor antagonists. J. Virol. 89, 9932–9938 (2015)

    Article  CAS  Google Scholar 

  20. Brecher, M. et al. Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change. J. Virol. 86, 364–372 (2012)

    Article  CAS  Google Scholar 

  21. Zhao, Y., Ren, J., Harlos, K. & Stuart, D. I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions. FEBS Lett. 590, 605–612 (2016)

    Article  CAS  Google Scholar 

  22. Jeffers, S. A., Sanders, D. A. & Sanchez, A. Covalent modifications of the ebola virus glycoprotein. J. Virol. 76, 12463–12472 (2002)

    Article  CAS  Google Scholar 

  23. Weissenhorn, W., Carfí, A., Lee, K. H., Skehel, J. J. & Wiley, D. C. Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol. Cell 2, 605–616 (1998)

    Article  CAS  Google Scholar 

  24. Gregory, S. M. et al. Structure and function of the complete internal fusion loop from Ebolavirus glycoprotein 2. Proc. Natl Acad. Sci. USA 108, 11211–11216 (2011)

    Article  ADS  CAS  Google Scholar 

  25. Flyak, A. I. et al. Cross-reactive and potent neutralizing antibody responses in human survivors of natural Ebolavirus infection. Cell 164, 392–405 (2016)

    Article  CAS  Google Scholar 

  26. Chandran, K., Sullivan, N. J., Felbor, U., Whelan, S. P. & Cunningham, J. M. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645 (2005)

    Article  ADS  CAS  Google Scholar 

  27. Dube, D. et al. The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding. J. Virol. 83, 2883–2891 (2009)

    Article  CAS  Google Scholar 

  28. Hood, C. L. et al. Biochemical and structural characterization of cathepsin L-processed Ebola virus glycoprotein: implications for viral entry and immunogenicity. J. Virol. 84, 2972–2982 (2010)

    Article  CAS  Google Scholar 

  29. Schornberg, K. et al. Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J. Virol. 80, 4174–4178 (2006)

    Article  CAS  Google Scholar 

  30. Hashiguchi, T. et al. Structural basis for Marburg virus neutralization by a cross-reactive human antibody. Cell 160, 904–912 (2015)

    Article  CAS  Google Scholar 

  31. Zhao, Y., Ren, J., Padilla-Parra, S., Fry, E. E. & Stuart, D. I. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor. Nat. Commun. 5, 4321 (2014)

    Article  ADS  CAS  Google Scholar 

  32. Desai, T. M. et al. IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion. PLoS Pathog. 10, e1004048 (2014)

    Article  Google Scholar 

  33. Cavrois, M., De Noronha, C. & Greene, W. C. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20, 1151–1154 (2002)

    Article  CAS  Google Scholar 

  34. Jones, D. M. & Padilla-Parra, S. Imaging real-time HIV-1 virion fusion with FRET-based biosensors. Sci. Rep. 5, 13449 (2015)

    Article  ADS  CAS  Google Scholar 

  35. Walter, T. S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D 61, 651–657 (2005)

    Article  Google Scholar 

  36. Winter, G., Lobley, C. M. & Prince, S. M. Decision making in xia2. Acta Crystallogr. D 69, 1260–1273 (2013)

    Article  CAS  Google Scholar 

  37. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

    Article  CAS  Google Scholar 

  38. Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003)

    Article  CAS  Google Scholar 

  39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  40. Stuart, D. I., Levine, M., Muirhead, H. & Stammers, D. K. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 A. J. Mol. Biol. 134, 109–142 (1979)

    Article  CAS  Google Scholar 

  41. DeLano, W. L. & Lam, J. W. PyMOL: A communications tool for computational models. Abstr. Pap. Am. Chem. Soc . 230, U1371–U1372 (2005)

    Google Scholar 

Download references

Acknowledgements

We thank Diamond scientists at I02 and I03 for assistance with data collection, T. S. Walter for help with crystallization and thermal-shift essay. Y.Z. was supported by the Biostruct-X project (283570) funded by the EU seventh Framework Programme (FP7), J.R. by the Wellcome Trust, and D.I.S., E.E.F. and K.H. by the UK Medical Research Council (MR/N00065X/1). This is a contribution from the UK Instruct Centre. The Wellcome Trust Centre for Human Genetics is supported by the Wellcome Trust (grant 090532/Z/09/Z). A.Z. is supported by a Marie Curie Fellowship (658363), T.A.B. is supported by the MRC (MR/L009528/1). SP-P is funded by a Nuffield Department of Medicine Leadership Fellowship. D.I.S. is a Jenner Investigator.

Author information

Author notes

  1. Yuguang Zhao and Jingshan Ren: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Structural Biology, University of Oxford, Wellcome Trust Centre for Human Genetics, Headington, OX3 7BN, Oxford, UK

    Yuguang Zhao, Jingshan Ren, Karl Harlos, Daniel M. Jones, Antra Zeltina, Thomas A. Bowden, Sergi Padilla-Parra, Elizabeth E. Fry & David I. Stuart

  2. Cellular Imaging Core, University of Oxford, Wellcome Trust Centre for Human Genetics, Headington, OX3 7BN, Oxford

    Sergi Padilla-Parra

  3. Diamond Light Source Ltd, Harwell Science &Innovation Campus, Didcot, OX11 0DE, UK

    David I. Stuart

Authors
  1. Yuguang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jingshan Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karl Harlos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel M. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Antra Zeltina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas A. Bowden
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sergi Padilla-Parra
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Elizabeth E. Fry
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David I. Stuart
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.Z., J.R. and D.I.S designed the project. Y.Z. made the protein and grew the crystals together with J.R., collected X-ray data and determined the structures. K.H. helped with crystal mounting and data collection. D.M.J. and S.P. carried out cell imaging experiments. A.Z. and T.A.B. provided the cDNA. Y.Z., J.R., E.E.F. and D.I.S. analysed the results and wrote the manuscript in discussions with all authors.

Corresponding author

Correspondence to David I. Stuart.

Ethics declarations

Competing interests

D.I.S., T.A.B. and A.Z. are listed as inventors on the International Patent Application No. PCT/GB2016/050321 ‘Filovirus therapy’.

Additional information

Reviewer Information Nature thanks E. Saphire, W. Weissenhorn and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Thermal-shift essay.

Representative thermal melt curves of EBOV GP with 10 μM compounds and 2% DMSO. a–d, Melting curves of EBOV GP with toremifene, ibuprofen or protein alone at pH 5.0, 6.0, 7.0 and 8.0, respectively. e, Small effects of SERM inhibitors tamoxifen, 4-hydroxytamoxifen and raloxifen on the melting temperature of EBOV GP shown at pH 5.2. f, Melt curves of EBOV GP with diaglycerol kinase inhibitor, anastrozole and benztropine mesylate at pH 5.2. g, h, Shifts in melting temperature (∆Tm °C in absolute value) were plotted against different concentrations of toremifene (g) or ibuprofen (h) at pH 5.2. Data are mean ± s.d. (n = 4). The affinity constant Kd is calculated by a ligand binding 1:1 saturation fitting with the SigmaPlot version 13 (Systat Software Inc.).

Extended Data Figure 2 Structural organization of EBOV GP and GP2 structure.

a, Scheme showing the structural organization of EBOV GP. FL, fusion loop; NHR and CHR, N- and C-terminal heptad repeats; SP, signal peptide; TM, transmembrane helix. The GPΔ construct used for structure determination is made by deleting residues 313–463 of the GP mucin domain and residues 633–676. Residue 312 is directly linked to 464. A foldon trimerization peptide and a 6× His tag are added at the C terminus. b, The GP2 trimer in the prefusion state (current structure). The trimer is shown as cartoon representation with the monomers coloured in red, green and blue, respectively. Disulfide bonds are shown as orange sticks. c, The six-helix bundle of GP2 in the post-fusion state.

Extended Data Figure 3 The fusion loop.

a, The fusion loop that connects β19 and β20 of GP2 projects onto a shallow depression on the surface of a neighbouring monomer. The fusion loop is shown as a red coil with side chains drawn as grey sticks, the neighbouring monomer is shown in semi-transparent surface representation. b, Comparison of the fusion loop in the apo GP (red and grey) obtained at pH 5.2 with that in the KZ52 Fab complex (cyan) obtained at pH 8.3.

Extended Data Figure 4 Pockets and tunnels in EBOV GP trimer.

a, The several small pockets and three large tunnels in the GP trimer shown as grey surfaces. Protein backbones are drawn as ribbons and coloured as in Fig. 2 of the main text. A toremifene is bound at the entrance of each large tunnel and shown as yellow sticks. b, Close up view of a tunnel. Each tunnel is bordered by secondary structure elements from two neighbouring monomers.

Extended Data Figure 5 The inhibitor-binding site.

a, The DFF lid (residues 192–194, blue coil for main chain and sticks for side chains) nestles at the entrance of the large tunnel in the apo structure. The rest of the protein is shown as an electrostatic surface. The putative cathepsin cleavage site at residue 190 is indicated by an arrow. b, c, Toremifene (yellow sticks in b) and ibuprofen (cyan sticks in c) bind at the same site by expelling the DFF lid. In both panels, the inhibitor bound structure is shown in blue (GP1) and red (GP2), the apo GP in grey. d, Comparing the binding modes of toremifene and ibuprofen. The toremifene-bound structure is shown in blue and red, the ibuprofen bound structure in grey.

Extended Data Figure 6 The environment of α1′ and α1 helices.

The surfaces of α1′ and α1 helices, which undergo large conformational changes upon receptor binding, are protected by the 287–293 loop from the glycan cap domain and the N563 glycan from GP2 in the apo GP. The glycan is modelled as Man9GlcNAc2.

Extended Data Figure 7 Chemical structures and electron density maps.

a, b, The chemical structures of toremifene (a) and ibuprofen (b). c, d, |Fo − Fc| omit electron density maps for toremifene (c) and ibuprofen (d) contoured at 3σ.

Extended Data Figure 8 Sequence alignment of filovirus GPs.

Amino acid sequence alignment of 7 filovirus GPs around the inhibitor-binding site. The amino acids that form contacts with toremifene or ibuprofen are coloured in green. Numbering corresponds to the full length Zaire EBOV GP, conserved residues are shown in a red background. Secondary structure elements are labelled on the top.

Extended Data Figure 9 Toremifene and ibuprofen inhibit fusion of Ebola GP pseudovirus particles.

a, CCF2-loaded TZM-bl cells were exposed to EBOV pseudoparticles (EBOVpp) or control particles lacking envelope proteins (NoENV) at 4 °C to synchronise binding and receptor engagement before fusion was initiated by shifting cells to 37 °C in the presence of toremefine (15 μM and 1.5 μM), ibuprofen (150 μM and 15 μM), or just the solvent (5% DMSO). After 2 h incubation, cells were loaded with the CCF2-AM FRET biosensor, fixed and the ratio of blue (440–480 nm, cleaved CCF2-AM) to green (500–540 nm, uncleaved CCF2-AM) fluorescence measured. Cells are pseudocoloured according to this ratio: blue represents no fusion, red represents fusion. Scale bar: 80 μm. b, The percentage of fusogenic cells (red versus blue) was calculated taking the average max value coming from the negative control as a threshold for fusion, data are means ± s.d. (n = 10). *P ≤ 0.05, ***P ≤ 0.001 (unpaired t-test, compared to the EBOV plus DMSO control). ns, not significant (P > 0.05). Error bars represent s.d.

Extended Data Table 1 Data collection and refinement statistics
Full size table

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, Y., Ren, J., Harlos, K. et al. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature 535, 169–172 (2016). https://doi.org/10.1038/nature18615

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature18615

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing