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Toremifene interacts with and destabilizes the Ebola virus glycoprotein


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.

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

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).


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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

Authors and Affiliations



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. ad, 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

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Zhao, Y., Ren, J., Harlos, K. et al. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature 535, 169–172 (2016).

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