Toremifene interacts with and destabilizes the Ebola virus glycoprotein

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 fusion. 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 deciphered. 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.

Extended Data Figure 1. Thermal shift essay.

Representative thermal melt curves of EBOV GP with 10 µM compounds and 2% DMSO. a-d, Melt curves of EBOV GP with toremifene, ibuprofen or protein alone at pH 5.0, pH 6.0, pH 7.0 and pH 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 pH5.2. g, h, Shifts in melting temperature (ΔTm °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 K_d 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. SP, signal peptide; FL, fusion loop; NHR and CHR, N- and C-terminal heptad repeats; TM, trans-membrane helix. The construct GPΔ 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. Disulphide bonds are drawn 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 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, |F_o-F_c| omit electron density maps for toremifene (c) and ibuprofen (d) contoured at 3σ.

Extended Data Figure 8. Sequence alignment of filovius 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 compounds solvent (5 % DMSO). After 2h 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, un-cleaved CCF2-AM) fluorescence measured. Cells are pseudocolored 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). Analysis used an unpaired t test and compared to the EBOV + DMSO control, where ns (not significant) = P > 0.05, * = P ≤ 0.05and *** = P ≤ 0.001. Error bars represent standard deviations.

Figure 1. Summary of thermal shift assays.

a, The effects of toremifene and ibuprofen on the melting temperature of EBOV GP at different pHs. The raw fluorescence traces are shown in Extended Data Fig. 1. Protein melting temperature at pH 5.2 at which crystals were grown is taken as the reference point. b, The melting temperatures of EBOV GP at different concentrations of toremifene or ibuprofen, at pH 5.2. Data are mean±s.d. (n=3).

Figure 2. Overall structure.

a, Cartoon diagram of EBOV GP monomer, GP1 blue, GP2 red and the glycan cap cyan. Secondary structural elements named as previously. Disulphide bonds shown as orange sticks, glycans in grey. The mucin domain omitted in our construct is shown as a yellow oval. FL, fusion loop. The C-terminal inserted foldon trimerization domain is disordered. b, The biological trimer viewed perpendicular to the 3-fold axis with one monomer coloured as in a and the second and third faded for clarity. c, The trimer viewed along the 3-fold, towards the viral membrane. d, Close up of the inhibitor binding site. e, Close up of the glycan cap and receptor binding site. Areas shown in d and e are indicated in panel a. In d and e antigenic sites are coloured grey and the receptor binding site yellow.

Figure 3. Structure comparisons.

a, Structure of the apo GP monomer compared with the GP of the KZ52 Fab complex. b-d, Details of the structural differences at the glycan cap (b), β1-β2 hairpin (c) and the CX₆CC motif (d). The apo GP is coloured as in Fig. 1a, the GP in complex with Fab grey. e, Comparison of apo GP with GP from the GP-receptor complex shown in same style as a. f-g, close up views of the major structural differences at α1’ and α1 helices (f), and β1-β2 hairpin (g).

Figure 4. Inhibitor binding site.

a, Details of the inhibitor biding site in the apo GP. The backbone is shown as ribbons with GP1 in blue and GP2 in red, side-chains as grey sticks. b, Tunnels of the GP trimer viewed along the 3-fold axis towards the viral membrane. Toremifenes bound at the entrances of the tunnels are shown as yellow sticks. c, Details of protein-inhibitor interactions of the GP-toremifene complex, and d, GP-ibuprofen complex. Toremifen is shown as yellow sticks, ibuprofen as cyan sticks. Protein main-chains are shown as ribbons and side-chains as sticks (GP1 blue, GP2 red). Side-chains in the apo structure with large conformational changes are shown as thinner grey sticks.