Structures of Ebola Virus Glycoprotein Complexes with Tricyclic

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 4938−4945

Structures of Ebola Virus Glycoprotein Complexes with Tricyclic Antidepressant and Antipsychotic Drugs Yuguang Zhao,†,# Jingshan Ren,†,# Elizabeth E. Fry,† Julia Xiao,‡ Alain R. Townsend,‡ and David I. Stuart*,†,§ †

Downloaded via UNIV OF EXETER on June 18, 2018 at 18:51:42 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, U.K. ‡ Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, U.K. § Diamond Light Source Ltd, Harwell Science & Innovation Campus, Didcot OX11 0DE, U.K. S Supporting Information *

ABSTRACT: A large number of Food and Drug Administration (FDA)-approved drugs have been found to inhibit the cell entry of Ebola virus (EBOV). However, since these drugs have various primary pharmacological targets, their mechanisms of action against EBOV remain largely unknown. We have previously shown that six FDA-approved drugs inhibit EBOV infection by interacting with and destabilizing the viral glycoprotein (GP). Here we show that antidepressants imipramine and clomipramine and antipsychotic drug thioridazine also directly interact with EBOV GP and determine the mode of interaction by crystallographic analysis of the complexes. The compounds bind within the same pocket as observed for other, chemically divergent complexes but with different binding modes. These details should be of value for the development of potent EBOV inhibitors.

■

INTRODUCTION Ebola virus (EBOV) bears a single protein on its outer membrane, GP, a type I fusion protein which mediates host cell attachment and endosome entry as well as membrane fusion.1−8 EBOV is internalized into host cells via macropinocytosis and subsequently trafficked through early and late endosomes. In the late endosome, EBOV GP engages with intracellular receptor NPC1, leading to a transfer of genome to the cytoplasm through membrane fusion.1,9−11 EBOV GP is therefore an obvious therapeutic target. EBOV is responsible for Ebola disease, which causes hemorrhagic fever and has a mortality rate of ∼50% in humans.12,13 There is currently no vaccine or therapeutic drug available. To shortcut the problematic process of drug development, a large number of FDA-approved drugs have been screened using either EBOV or pseudotyped virus assays.14−22 The number of drugs that inhibit EBOV infection or pseudotyped virus entry is surprisingly large; however, the precise mechanism of inhibition is largely unknown. We have previously demonstrated that six such drugs interact directly with and decrease the thermal stability of EBOV GP and have determined their crystal complex structures.23,24 Although these six compounds have varied chemical structures and five different primary pharmaco© 2018 American Chemical Society

logical targets (Figure S1), all of them bind within the same cavity. Since the drug−binding cavity is large, the potential protein−inhibitor interactions are far from fully exploited. Here we report that three tricyclic drugs, the antidepressants imipramine and clomipramine and antipsychotic thioridazine (Figure 1), also bind EBOV GP and present crystal structures of these three drugs binding within this same cavity.

Figure 1. Chemical structures. (A) Imipramine, 3-(5,6-dihydrobenzo[b][1]benzazepin-11-yl)-N,N-dimethylpropan-1-amine. (B) Clomipramine, 3-(2-chloro-5,6-dihydrobenzo[b][1]benzazepin-11-yl)-N,Ndimethylpropan-1-amine. (C) Thioridazine, 10-[2-(1-methylpiperidin-2-yl)ethyl]-2-methylsulfanylphenothiazine. Received: March 4, 2018 Published: May 9, 2018 4938

DOI: 10.1021/acs.jmedchem.8b00350 J. Med. Chem. 2018, 61, 4938−4945

Journal of Medicinal Chemistry

Article

Table 1. Binding Affinity to EBOV GP and Inhibition Activity.a IC50 (μM) imipramine clomipramine thioridazine toremifene

ΔTm (°C)

Kd (μM)

E-S-Flu VLP (MDCK-SIA1 cells)

eGFP-EBOV (vero cells)b

−2 −3

584 (256) 118 (40)

13.2 (0.3) 10.3 (0.3) 7.8 (2.3) 0.15e

11.4 (0.15) 6.24 (0.79) 0.162 (0.048)

−15

16 (4)d

ebola VLP (hela cells)c 13.7 4.99 0.566

a

Values adapted. The standard deviation is shown in parentheses. Toremifene is included for comparison. bJohansen et al.15 cKouznetsova et al.16 d Zhao et al.24 eXiao et al.22

Figure 2. Overall structure of Ebola GP and electron density maps. (A) The trimeric structure of EBOV GP (PDB ID 6G95); GP1 is in blue, GP2 is in red, and the glycan cap is in cyan. The bound thioridazine at the entrance of a tunnel is shown as orange spheres. (B) Close-up of the EBOV GP drug binding pocket in a surface representation. Structures of GP−toremifene (PDB ID 5JQ7), GP−bepridil (PDB ID 6F5U), and GP− clomipramine (PDB ID 6G9I) are first overlapped with GP−thioridazine (PDB ID 6G95) and then the positions and orientations of the bound inhibitors in the pocket are shown as sticks (yellow, magenta, cyan, and orange, respectively). (C−E) Simulated annealing |Fo−Fc| omit electron density maps for imipramine (C), clomipramine (D), and thioridazine (E) contoured at 3.5σ. The red density in (C) is contoured at 5σ, showing that one imipramine molecule is less well ordered.

■

RESULTS AND DISCUSSION Identification of GP−Drug Interaction. Imipramine, clomipramine, and thioridazine have been reported to inhibit both EBOV infection and EBOV−virus-like particle (VLP) entry into cells.15,16,25 We performed thermal shift assays to determine if these compounds altered the thermal stability of EBOV GP since the perturbation of thermal stability indicates direct interaction.26 The experiment was carried out at pH 5.2, which is close to the physiological pH of the late endosome where fusion takes place and also the pH at which the EBOV GP crystals were grown. The results show that imipramine and clomipramine both decrease the melting temperature (Tm) of EBOV GP by 2 °C at 500 μM concentration, which is much smaller than the 15 °C by toremifene24 and 6 °C by bepridil but similar in magnitude to that due to sertraline.23 The binding

constants (Kd values) derived from thermal shift assays are 0.58 mM for imipramine and 0.12 mM for clomipramine (Figure S2). Since thioridazine interacts directly with the dye (SYPRO Orange) used in the assay, its effect on the melting temperature of EBOV GP cannot be determined by this method; however, the soaking of GP crystals with thioridazine showed that thioridazine interacts directly with EBOV GP (see below). Inhibition of EBOV GP Pseudotyped Flu Virus Entry. To determine the antiviral activity of these three drugs we performed a cell entry inhibition assay exactly as described by Xiao et al.22 E-S-FLU is an influenza virus core pseudotyped (coated) with Zaire Ebola glycoprotein (GenBank KJ660346.1). The concentration of small molecules required to give 50% reduction in infection, which is measured in triplicate, is 13.2 ± 0.3 μM for imipramine, 10.3 ± 0.3 μM for 4939



Article

clomipramine, and 7.8 ± 2.3 μM for thioridazine (Table 1). The values are in good agreement with those obtained using the infectious virus15 or a different type of Ebola VLP.16 Structure Determination. The structures of EBOV GP− drug complexes were obtained by soaking crystals of GP in drug-containing solutions (Experimental Section). Highly redundant X-ray diffraction data sets, extending to 2.35 Å resolution or better, were collected on beamlines of the Diamond Light Source synchrotron (see Experimental Section). The structures were determined using molecular replacement and refined to reasonable R factors with good stereochemistry (Table S1). The electron density maps for the bound clomipramine and thioridazine allowed the chemical groups of the drugs to be modeled unambiguously. However, two molecules of imipramine (named A and B thereafter) are bound, and while one has well-defined electron density, the second is less ordered (Figure 2). The overall protein structures of these three GP−drug complexes are very similar to each other as well as to the previously published structures of GP and GP−drug complexes.23,24,27,28 Taking the GP−bepridil complex (the highest resolution of those we have determined previously) as a reference, GP−imipramine, GP−clomipramine, and GP−thioridazine have 376, 376, and 379 (out of 382) Cα values, matching with rmsd’s of 0.45, 0.48, and 0.48 Å, respectively. Residues 46−52 preceding the disulfide bond (C53−C609) that links GP1 and GP2 have two conformations in the GP−imipramine and GP−thioridazine complexes. This was previously observed in the GP−paroxetine complex and does not appear to be related to inhibitor binding. Residues 521−525 preceding the fusion loop and the C-terminal helix in GP2 have relatively weaker electron density, indicative of flexibility. Characteristics of the Drug-Binding Site. The structures of EBOV GP reported here are composed of GP1 and GP2 subunits and are in the prefusion state. Three copies of the GP form the biological trimer around a 3-fold crystallographic axis. In each GP monomer the two subunits are linked by a disulfide and GP2 wraps in a semicircle around the N-terminal end of GP1. The receptor binding site is located in GP1 and protected by a glycan cap (Figure 2). In the late endosome/lysosome cathepsin, B/L removes the glycan cap to allow for the binding of the receptor,9,10 which subsequently triggers the uncoupling of GP2 from GP1. GP2 then undergoes large conformation changes which ultimately lead to membrane fusion and the radically different postfusion state.29,30 In our apo prefusion structure, each monomer harbors a tunnel, and the three tunnels of the trimer join at the three-fold axis. The tunnel entrance, located between GP1 and GP2, is blocked by a tight turn called the DFF lid (residues 192−194). Inhibitors, including the three described here, bind at the tunnel entrance by expelling the DFF lid.23,24 The inhibitor binding cavity is large, having a volume of approximately 1000 Å3, and sits directly underneath the stem of the fusion loop, some 30 Å from its tip. The distances from the inhibitor binding site to the receptor binding site and the viral membrane are about 35 and 65 Å, respectively (Figure 2). The internal surface of the cavity is largely hydrophobic, apart from the area at the mouth of the tunnel that is surrounded by charged or hydrophilic residues. Residues involved in inhibitor binding are contributed by the β1−β2 hairpin, β3, β6, and β13 of GP1, the stem of the fusion loop (β19−β20), and α3 of GP2. All nine drugs that inhibit EBOV by directly interacting with the GP bind in this same cavity.23,24 The molecular volume of

the drugs ranges from ibuprofen, the smallest at 188 Å3, to toremifene, the largest at 362 Å3, and when superposed they span a total volume of 878 Å3 (excluding the less ordered benztropine B and imipramine B), of which only 14 Å3 is common to all nine drugs (Figure S3). The drug−protein interactions are almost entirely hydrophobic; the only hydrogen bond observed is from the propanoic acid moiety of ibuprofen to the side chain of R64.24 Different inhibitors induce subtle but different conformational changes at the binding site. Interactions of EBOV GP with Imipramine. Two imipramine molecules bind in the cavity, one in front of M548 and the other close to Y517 (Figures 2C and 3A).

Figure 3. Binding of imipramine. (A) Two imipramine molecules (gray sticks) are bound in the binding pocket (PDB ID 6G9B). Protein main chains are shown as ribbons, and the side chains are shown as sticks. The color scheme is as in Figure 2. Side chains with large conformational changes in the apo GP structure are drawn as thinner gray sticks. (B) Comparison of the positions and orientations of the imipramines (gray sticks) and benztropines (PDB ID 6F6S; green sticks) in the binding pocket. F193 and F194, which occupy the binding site in the apo GP (PDB ID 5JQ3), are shown as red sticks.

Imipramine has two benzene rings fused to a seven-membered azepine group (Figure 1). Molecule A has well defined electron density and binds in the volume occupied by F193 in the apo GP, with its azepine ring nestling in a subpocket adjacent to α3. One benzene ring has interactions with the side chains of L186, L515, M548, and L558; the second is flanked by I38 and L184 and also contacts L43 and L186. The dimethylpropanamine side chain points toward the solvent and makes no contact (≤3.9 Å) with protein atoms (Figure 3). Molecule B of imipramine binds in the volume occupied by F194 in apo GP, sandwiched between the side chains of R64 and Y517. One benzene ring has T-shaped stacking interactions with both Y517 and a benzene ring of molecule A and hydrophobic contacts with A101 and M548; the second benzene ring and the dimethylpropanamine group are solvent-exposed with weaker electron density and have no strong protein interactions (Figures 3 and S4). 4940



Article

with that of the corresponding benzene ring of molecule A of imipramine. However, clomipramine has better Kd and IC50 values, demonstrating that occupying the FF volume is pivotal for inhibitor binding (Figures 4 and S4). Unique Binding Mode of Thioridazine. Thioridazine has a dibenzthiazine tricyclic ring instead of the dibenzazepine present in imipramine and clomipramine. Intriguingly, thioridazine binds in the cavity in a dramatically different mode (Figure 5). Its dibenzthiazine is rotated ∼110° from the

The volume in front of Y517 and M548 is occupied by F193 and F194 in the apo structure and is termed the FF volume. We have previously shown that this volume has a propensity to be occupied, presumably stabilizing the hydrophobic residues around it. Among all nine complexes we have now determined (including the three new structures described here) that ibuprofen and sertraline are the smallest, and both occupy this volume. In the case of benztropine, two molecules are required to fill the FF volume (Figure 3B), and paroxetine partially fills the volume, with an unidentified small molecule or a less ordered paroxetine filling the remainder.23,24 The two imipramine molecules bind in the cavity with the dibenzazepines oriented and positioned similarly to the two phenyl rings of the two bound benztropine molecules, although the centers of the two imipramine molecules shift 1.2 Å toward the β1−β2 hairpin, presumably due to the greater bulk and rigidity of the dibenzazepine rings (Figure 3). Binding of Clomipramine. The difference between clomipramine and imipramine, merely a chlorine atom on one of the benzene rings, is sufficient to switch the binding mode so that only a single molecule of clomipramine is bound. The bound clomipramine roughly overlaps with molecule A of imipramine but is shifted 0.8 Å toward molecule B of imipramine such that the chlorine group is positioned in front of Y517, increasing the overlap with the FF volume compared to that of molecule A of imipramine (Figure 4). The chlorine atom does not make any contact with the protein atoms, and the benzene ring makes no contact with I185 and L515 and has fewer interactions with I38 and L43 compared

Figure 5. Binding of thioridazine. (A) Thioridazine (PDB ID 6G95; orange sticks) in the binding pocket. Protein main chains are shown as ribbons, and side chains are shown as sticks. The color scheme is as in Figure 2. Side chains with large conformational changes in the apo GP structure are drawn as thinner gray sticks. (B) Comparison of the binding modes of thioridazine and clomipramine (PDB ID 6G9I; cyan sticks). Protein main chains and side chains associated with thioridazine are shown as ribbons and sticks and are shown in atom colors, and the side chains of the GP-clomipramine complex are shown as thinner gray sticks.

dibenzazepine of clomipramine, positioning the methylsulfanyl group between the side chains of I38 and L184 and the benzene ring between M548 and α3, spaces not exploited by any other bound drugs. The 2-ethyl-1-methylpiperidine group folds down to the cavity in front of Y517, overlapping with the chlorobenzene group of the clomipramine rather than the corresponding dimethylpropanamine group of clomipramine which points up to the solvent. The methylsulfanylbenzene group partially overlaps the benzene ring of clomipramine, with the methylsulfanyl moiety undergoing extensive interactions with the side chains of I38, L43, and L186 as well as the mainchain atoms of V37 and L186; the other benzene ring contacts the side chains of M548 and I555 and the carbonyl oxygen of H549 (Figures 5 and S4). Thioridazine occupies the key FF volume and has more protein interactions, explaining why it is the strongest of the inhibitors reported here.

Figure 4. Binding of clomipramine. (A) Clomipramine (PDB ID 6G9I; cyan sticks) in the binding pocket. Protein main chains are shown as ribbons, and side chains are shown as sticks. The color scheme is as in Figure 2. Side chains with large conformational changes in the apo GP (PDB ID 5JQ3) structure are drawn as thinner gray sticks. (B) The comparison of the binding modes of clomipramine and imipramine (PDB ID 6G9B). Protein main chains and side chains are those associated with clomipramine. 4941



Article

Figure 6. Comparison with the binding mode of toremifene. The structure of GP−toremifene (PDB ID 5JQ7) is overlapped with GP− clomipramine (PDB ID 6G9I) (A), and their fit in the cavity is shown as an electrostatic surface (B). The structure of GP−toremifene is overlapped with GP−thioridazine (PDB ID 6G95) (C) and shows their fit in the cavity (the protein is shown as an electrostatic surface) (D). In panels (A,C), toremifene is shown as yellow sticks, and the side chains of its associated GP are shown as thinner gray sticks where there are significant conformational changes.

■

Implications for Drug Design. Driven by their chemical diversity, the nine inhibitors now characterized bind in varied positions and orientations in the cavity, exploring different protein interactions. Drug binding is achieved by shape complementarity, facilitated by conformational changes in the protein residues, while affinity arises mainly from hydrophobic interactions. The nine inhibitors between them sample 88% of the cavity volume (Figure S3). However, the volume exploited by each inhibitor is small, and some areas of the binding site remain poorly explored, for example, the mouth of the tunnel where there are several charged/polar residues, including R64, E100, T519, and D522. Modifications could be made to some drugs such as toremifene and bepridil by introducing a group to make hydrogen bonding interactions which might substantially enhance the binding affinity, e.g., replacing the dimethylamine moiety of toremifene with a carboxamide group to form a hydrogen bond with the side chain of D522. Toremifene is the most potent inhibitor characterized to date, with an IC50 that is 50-fold better than for clomipramine and thioridazine and has little overlap with the latter two (Figure 6). A simple substitution of the chlorine atom of toremifene by a benzyl ring or the methylsulfanylbenzene group of thioridazine might greatly improve its potency. In addition, a piperidine or piperazine substitution of ring A of toremifene that binds in a subpocket adjacent to V66 and A101 with a negatively charged electrostatic surface at the bottom would likely be more optimal for binding (Figures 6 and S1). Perhaps a chimeric molecule designed from the well-fitted fragments of several inhibitors might substantially improve the potency beyond that of those identified to date.

CONCLUSIONS The number of FDA-approved drugs that have been found to inhibit Ebola virus entry is astonishing. These drugs have various primary pharmacological targets, huge chemical diversity, and probably varied inhibition mechanisms. Nevertheless, we have now demonstrated that nine of these drugs directly interact with EBOV GP by using a thermal shift assay and crystal soaking and have determined their complex structures with the GP. All nine bind at the same site, and eight decrease the thermal stability of the protein (thermal stability could not be determined in the presence of thioridazine); the binding affinity, protein−drug interactions, and IC50 values correlate reasonably well, strongly suggesting that they inhibit EBOV entry via the same mechanism. We have proposed that inhibitor binding destabilizes GP and triggers the premature release of GP2, thereby preventing fusion between the viral and endosome membranes.24 The DFF lid is positioned immediately after the putative cathepsin B/L cleavage site 31−35 and may function to maintain the conformation of the cleavage site. Inhibitor binding expels the DFF lid from the pocket, therefore altering the conformation of the cleavage site. Thus, an alternative mechanism of inhibition could be that inhibitor binding renders cathepsin B/L unable to remove the glycan cap domain, preventing the binding of receptor NPC1. Protein residues lining the drug-binding cavity are highly conserved among the five known species of EBOVs; therefore, we expect the nine drugs to bind the GPs of all EBOV species. Our fully refined high-resolution structures reveal the basis for specificity for these inhibitors, which can guide the design of more potent inhibitors to combat Ebola disease. 4942



■

Article

on solubility. Eight crystals were soaked for each inhibitor in the above solutions for different lengths of time, ranging from 2 to 20 min. X-ray Data Collection. The soaked crystals were transferred to solutions containing 75% inhibitor soaking solution and 25% (v/v) glycerol for a couple of seconds and then frozen in liquid nitrogen prior to data collection. All data were collected at 100 K with a frame of 0.1° rotation using synchrotron X-rays and PILATUS 6 M detectors at Diamond Light Source, U.K. GP-imipramine and GP-clomipramine data were acquired on beamline I04 with a beam size of 63 × 50 μm2 and a wavelength of 0.9795 Å. The exposure time per data frame was 0.1 or 0.12 s with 100% beam transmission. GP−thioridazine data were collected on beamline I24 with a beam size of 50 × 50 μm2 and a wavelength of 0.9686 Å. The exposure time per data frame was 0.01 s with 50% beam transmission (since I24 has a stronger beam, the shortest possible exposure time per frame was used).38,39 Data were collected over 360° from every crystal that diffracted. Eight out of eight crystals soaked with imipramine or thioridazine and three out of eight crystals soaked with clomipramine diffracted. Data Processing, Structure Determination, and Structure Refinement. Diffraction images were indexed, integrated, and scaled with the Xia2 automated data processing program using the 3dii or Dials protocols.40,41 Each data set was initially phased with rigid-body refinement using the apo GP structure (PDB ID 5JQ3), omitting residues 190−195 of GP1 and water molecules. The electron density maps calculated at this stage were carefully checked. Only those data sets that give the best electron density for the soaked drugs were used for the later structure refinement. Thus, the final data set for GP− imipramine is from a single crystal, while the GP−clomipramine and GP−thioridazine complexes use two crystals each. The resolution of the diffraction data for the three complexes ranges from 2.23 to 2.31 Å. Structure refinement used REFMAC542 or PHENIX,43 and models were rebuilt with COOT.44 All three structures were refined to reasonable R factors with good stereochemistry. Data collection and structure refinement statistics are given in Table S1. Structural comparisons used SHP.45 Simulated annealing omitted electron density maps were calculated with CNS,46 volumes of the drugbinding cavity and drug molecules were calculated with VOLUMES (Robert Esnouf, unpublished data), and figures were prepared with PyMOL47 and LigPlot.48

EXPERIMENTAL SECTION

Protein Expression and Purification. The construct of the EBOV (Zaire strain Mayinga−76) glycoprotein extracellular domain as described earlier24 with one extra H613A mutation was cloned in the mammalian expression vector pNeosec.36 The resulting plasmid pNeosec−GPΔ has the mucin domain deleted and is tagged with a foldon trimerization sequence from the bacteriophage T4 fibritin and six histidines at the C terminus. The endotoxin-free plasmid was transiently transfected into human embryonic kidney HEK293T (ATCC CRL11268) cells with polyethylenimine (MW 25kD, Sigma, U.K.). The transfection mixture was further supplemented with the mannosidase inhibitor kifunensine (Cayman Chemical, Ann Arbor, MI, USA) at a final concentration of 5 μM to limit the protein glycosylation and facilitate crystallization. The conditioned medium was collected 5 days after transfection and dialyzed against PBS. The His-tagged protein was captured with talon beads (Takara Bio Europe SAS, France) at 15 °C for 1 h with gentle shaking at 110 rpm. The beads were collected and washed in PBS with 5−10 mM imidazole. The protein was eluted with 200 mM imidazole in PBS and further purified by size-exclusion chromatography with a Superdex 200 HiLoad 16/600 column (GE Healthcare, Buckinghamshire, U.K.) and a buffer of 10 mM MES, pH 5.2, and 150 mM NaCl. Reagents. Imipramine hydrochloride (Sigma−I0899) with specified purity of ≥99% and clomipramine hydrochloride (Sigma−C7291) with specified purity of ≥98% were purchased from Sigma-Aldrich. Thioridazine hydrochloride (KS−5108) with specified purity of ≥97% was purchased from Key Organics. Thermal Shift Assay. Each compound was initially dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted (1:10) with a buffer of 25 mM sodium citrate at pH 5.2 and 150 mM NaCl. Then the compounds were subjected to a 2-fold serial dilution to the desired concentration with 10% DMSO of the above buffer. Twenty-five microliters each of diluted compounds in a semiskired 96-well PCR plate was mixed with an equal amount of 2 μM freshly purified glycosylated EBOV GP protein in a buffer of 25 mM sodium citrate at pH 5.2, 150 mM NaCl, and 6× SYPRO Orange dye (Thermo Fisher Scientific, U.K.). The samples were then heated in an Mx3005p qPCR machine (Stratagene, Agilent Technologies, USA) from room temperature at a rate of 1 °C min−1 for 74 cycles. Fluorescence changes were monitored with excitation and emission wavelengths at 492 and 610 nm, respectively. Reference wells, i.e., solutions without drugs but with the same amount of DMSO, were used to compare the melting temperature (Tm). Experiments were carried out in triplicate. Inhibition Assay of E-S-FLU. E-S-FLU is a single-cycle influenza virus with the hemagglutinin coding sequence replaced with eGFP and pseudotyped (coated) with the Ebola glycoprotein.22 Infection is dependent on the expression of the NPC-1 receptor for Ebola. Infected MDCK-SIAT1 indicator cells fluoresce brightly, and the virus was titrated to give saturating infection. Inhibitors were cultured with the cells before the addition of E-S-FLU, and the concentration of drug required to suppress infection by 50% was measured in triplicate by linear interpolation as described in Xiao et al.22 Crystallization and Inhibitor Soaking. Crystallization of EBOV GP was carried out using the sitting-drop vapor-diffusion method as described previously.24 Crystals were grown in conditions containing 9% (w/v) PEG 6000 and 0.1 M sodium citrate tribasic dihydrate at pH 5.2. The crystallization drop is composed of 100 nL protein solution at a concentration of 10−12 mg/mL buffered in 10 mM MES at pH 5.2 and 100 nL microseed solution that was prepared by crushing a crystal of about 50 × 50 × 50 μm3 in size in 50 μL of reservoir solution by vortex mixing with a Teflon bead and then further diluting 50× with reservoir solution.37 Crystallization of EBOV GP with microcrystal seeds normally gave reasonably sized crystals in most drops. GP and inhibitor complexes were obtained by crystal soaking experiments. The crystal soaking solutions were prepared by first dissolving the inhibitors in 100% DMSO and then diluting the dissolved inhibitors in 15% (w/v) PEG 6000 and 0.1 M sodium citrate tribasic dihydrate (pH 5.2) to a final DMSO concentration of 10%. The inhibitor concentration was typically from 1 to 10 mM depending

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00350. Figures S1−S4, which show the chemical structures of drugs whose structures with EBOV GP have been reported previously, summaries of thermal shift assay data, the molecular volumes of the bound drugs, and diagrams showing protein−drug interactions. Table S1 shows X-ray data and structure refinement statistics. (PDF) SMILES (CSV) Accession Codes

The coordinates and structure factors have been deposited with the RCSB Protein Data Bank under accession codes 6G9B, 6G9I, and 6G95 for GP−imipramine, GP−clomipramine, and GP−thioridazine, respectively, and will be released upon article publication.

■

AUTHOR INFORMATION

Corresponding Author

*Fax: +44 (0)1865 287501. E-mail: [email protected]. ORCID

Jingshan Ren: 0000-0003-4015-1404 4943



Article

(10) 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. 2016, 590, 605−612. (11) Gong, X.; Qian, H.; Zhou, X.; Wu, J.; Wan, T.; Cao, P.; Huang, W.; Zhao, X.; Wang, X.; Wang, P.; Shi, Y.; Gao, G. F.; Zhou, Q.; Yan, N. Structural insights into the Niemann-Pick C1 (NPC1)-mediated cholesterol transfer and Ebola infection. Cell 2016, 165, 1467−1478. (12) Anthony, S. M.; Bradfute, S. B. Filoviruses: One of these things is (not) like the other. Viruses 2015, 7, 5172−5190. (13) Coltart, C. E.; Lindsey, B.; Ghinai, I.; Johnson, A. M.; Heymann, D. L. The Ebola outbreak, 2013−2016: old lessons for new epidemics. Philos. Trans. R. Soc., B 2017, 372, 1−24. (14) Johansen, L. M.; Brannan, J. M.; Delos, S. E.; Shoemaker, C. J.; Stossel, A.; Lear, C.; Hoffstrom, B. G.; Dewald, L. E.; Schornberg, K. L.; Scully, C.; Lehar, J.; Hensley, L. E.; White, J. M.; Olinger, G. G. FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Sci. Transl. Med. 2013, 5, 190ra179. (15) Johansen, L. M.; DeWald, L. E.; Shoemaker, C. J.; Hoffstrom, B. G.; Lear-Rooney, C. M.; Stossel, A.; Nelson, E.; Delos, S. E.; Simmons, J. A.; Grenier, J. M.; Pierce, L. T.; Pajouhesh, H.; Lehar, J.; Hensley, L. E.; Glass, P. J.; White, J. M.; Olinger, G. G. A screen of approved drugs and molecular probes identifies therapeutics with anti-Ebola virus activity. Sci. Transl. Med. 2015, 7, 290ra89. (16) Kouznetsova, J.; Sun, W.; Martinez-Romero, C.; Tawa, G.; Shinn, P.; Chen, C. Z.; Schimmer, A.; Sanderson, P.; McKew, J. C.; Zheng, W.; Garcia-Sastre, A. Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerging Microbes Infect. 2014, 3, e84. (17) Yermolina, M. V.; Wang, J.; Caffrey, M.; Rong, L. L.; Wardrop, D. J. Discovery, synthesis, and biological evaluation of a novel group of selective inhibitors of filoviral entry. J. Med. Chem. 2011, 54, 765−781. (18) Edwards, M. R.; Pietzsch, C.; Vausselin, T.; Shaw, M. L.; Bukreyev, A.; Basler, C. F. High-throughput minigenome system for identifying small-molecule inhibitors of Ebola virus replication. ACS Infect. Dis. 2015, 1, 380−387. (19) Anantpadma, M.; Kouznetsova, J.; Wang, H.; Huang, R.; Kolokoltsov, A.; Guha, R.; Lindstrom, A. R.; Shtanko, O.; Simeonov, A.; Maloney, D. J.; Maury, W.; LaCount, D. J.; Jadhav, A.; Davey, R. A. Large-scale screening and identification of novel Ebola virus and Marburg virus entry inhibitors. Antimicrob. Agents Chemother. 2016, 60, 4471−4481. (20) Basu, A.; Li, B.; Mills, D. M.; Panchal, R. G.; Cardinale, S. C.; Butler, M. M.; Peet, N. P.; Majgier-Baranowska, H.; Williams, J. D.; Patel, I.; Moir, D. T.; Bavari, S.; Ray, R.; Farzan, M. R.; Rong, L.; Bowlin, T. L. Identification of a small-molecule entry inhibitor for filoviruses. J. Virol 2011, 85, 3106−3119. (21) Cheng, H.; Schafer, A.; Soloveva, V.; Gharaibeh, D.; Kenny, T.; Retterer, C.; Zamani, R.; Bavari, S.; Peet, N. P.; Rong, L. Identification of a coumarin-based antihistamine-like small molecule as an antifiloviral entry inhibitor. Antiviral Res. 2017, 145, 24−32. (22) Xiao, J.; Rijal, P.; Schimanski, L.; Tharkeshwar, A. K.; Wright, E.; Annaert, W.; Townsend, A. Characterization of an influenza virus pseudotyped with Ebolavirus glycoprotein. J. Virol. 2018, 92, JVI.00941−17. (23) Ren, J.; Zhao, Y.; Fry, E. E.; Stuart, D. I. Target identification and mode of action of four chemically divergent drugs against Ebola virus infection. J. Med. Chem. 2018, 61, 724−733. (24) Zhao, Y.; Ren, J.; Harlos, K.; Jones, D. M.; Zeltina, A.; Bowden, T. A.; Padilla-Parra, S.; Fry, E. E.; Stuart, D. I. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature 2016, 535, 169−172. (25) Miller, M. E.; Adhikary, S.; Kolokoltsov, A. A.; Davey, R. A. Ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection. J. Virol 2012, 86, 7473−7483. (26) Walter, T. S.; Ren, J.; Tuthill, T. J.; Rowlands, D. J.; Stuart, D. I.; Fry, E. E. A plate-based high-throughput assay for virus stability and vaccine formulation. J. Virol. Methods 2012, 185, 166−170.

David I. Stuart: 0000-0002-3426-4210 Author Contributions #

These authors contributed equally to this work. Y.Z., J.R., and D.I.S designed the project. Y.Z., J.R., and J.X. performed experiments. J.R., Y.Z., and D.I.S. analyzed the results together with E.E.F. and J.X., and A.R.T., J.R., and D.I.S. wrote the manuscript in discussion with all authors. All authors read and approved the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank Diamond Light Source for beam time (proposal mx10627) and the staff of beamlines I04 and I24 for assistance with crystal testing and data collection. 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. and E.E.F. by the U.K. Medical Research Council (MR/N00065X/1). This is a contribution from the U.K. Instruct Centre, part of Instruct-ERIC. The Wellcome Centre for Human Genetics is supported by Wellcome (grant 090532/Z/09/Z).

■

ABBREVIATIONS USED DMSO, dimethyl sulfoxide; EBOV, Ebola virus; FDA, Food and Drug Administration; GP, glycoprotein; VLP, virus-like particle

■

REFERENCES

(1) Carette, J. E.; Raaben, M.; Wong, A. C.; Herbert, A. S.; Obernosterer, G.; Mulherkar, N.; Kuehne, A. I.; Kranzusch, P. J.; Griffin, A. M.; Ruthel, G.; Dal Cin, P.; Dye, J. M.; Whelan, S. P.; Chandran, K.; Brummelkamp, T. R. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 2011, 477, 340− 343. (2) Hacke, M.; Bjorkholm, P.; Hellwig, A.; Himmels, P.; de Almodovar, C. R.; Brugger, B.; Wieland, F.; Ernst, A. M. Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol. Nat. Commun. 2015, 6, 7688. (3) Nanbo, A.; Imai, M.; Watanabe, S.; Noda, T.; Takahashi, K.; Neumann, G.; Halfmann, P.; Kawaoka, Y. Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog. 2010, 6, e1001121. (4) 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. 2010, 6, e1001110. (5) Takada, A.; Robison, C.; Goto, H.; Sanchez, A.; Murti, K. G.; Whitt, M. A.; Kawaoka, Y. A system for functional analysis of Ebola virus glycoprotein. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 14764− 14769. (6) Aleksandrowicz, P.; Marzi, A.; Biedenkopf, N.; Beimforde, N.; Becker, S.; Hoenen, T.; Feldmann, H.; Schnittler, H. J. Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis. J. Infect. Dis. 2011, 204 (Suppl_3), S957−967. (7) Hunt, C. L.; Kolokoltsov, A. A.; Davey, R. A.; Maury, W. The Tyro3 receptor kinase Axl enhances macropinocytosis of Zaire ebolavirus. J. Virol 2011, 85, 334−347. (8) Mulherkar, N.; Raaben, M.; de la Torre, J. C.; Whelan, S. P.; Chandran, K. The Ebola virus glycoprotein mediates entry via a nonclassical dynamin-dependent macropinocytic pathway. Virology 2011, 419, 72−83. (9) Wang, H.; Shi, Y.; Song, J.; Qi, J.; Lu, G.; Yan, J.; Gao, G. F. Ebola Viral glycoprotein bound to its endosomal receptor Niemann-Pick C1. Cell 2016, 164, 258−268. 4944



Article

(27) Lee, J. E.; Fusco, M. L.; Hessell, A. J.; Oswald, W. B.; Burton, D. R.; Saphire, E. O. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 2008, 454, 177−182. (28) Dias, J. M.; Kuehne, A. I.; Abelson, D. M.; Bale, S.; Wong, A. C.; Halfmann, P.; Muhammad, M. A.; Fusco, M. L.; Zak, S. E.; Kang, E.; Kawaoka, Y.; Chandran, K.; Dye, J. M.; Saphire, E. O. A shared structural solution for neutralizing ebolaviruses. Nat. Struct. Mol. Biol. 2011, 18, 1424−1427. (29) Malashkevich, V. N.; Schneider, B. J.; McNally, M. L.; Milhollen, M. A.; Pang, J. X.; Kim, P. S. Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-A resolution. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2662−2667. (30) Weissenhorn, W.; Carfi, 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 1998, 2, 605− 616. (31) 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 2005, 308, 1643−1645. (32) Flyak, A. I.; Shen, X.; Murin, C. D.; Turner, H. L.; David, J. A.; Fusco, M. L.; Lampley, R.; Kose, N.; Ilinykh, P. A.; Kuzmina, N.; Branchizio, A.; King, H.; Brown, L.; Bryan, C.; Davidson, E.; Doranz, B. J.; Slaughter, J. C.; Sapparapu, G.; Klages, C.; Ksiazek, T. G.; Saphire, E. O.; Ward, A. B.; Bukreyev, A.; Crowe, J. E., Jr. Crossreactive and potent neutralizing antibody responses in human survivors of natural Ebolavirus infection. Cell 2016, 164, 392−405. (33) Dube, D.; Brecher, M. B.; Delos, S. E.; Rose, S. C.; Park, E. W.; Schornberg, K. L.; Kuhn, J. H.; White, J. M. The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding. J. Virol 2009, 83, 2883−2891. (34) Hood, C. L.; Abraham, J.; Boyington, J. C.; Leung, K.; Kwong, P. D.; Nabel, G. J. Biochemical and structural characterization of cathepsin L-processed Ebola virus glycoprotein: implications for viral entry and immunogenicity. J. Virol 2010, 84, 2972−2982. (35) Schornberg, K.; Matsuyama, S.; Kabsch, K.; Delos, S.; Bouton, A.; White, J. Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J. Virol 2006, 80, 4174−4178. (36) Zhao, Y.; Ren, J.; Padilla-Parra, S.; Fry, E. E.; Stuart, D. I. Lysosome sorting of beta-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor. Nat. Commun. 2014, 5, 4321. (37) Walter, T. S.; Mancini, E. J.; Kadlec, J.; Graham, S. C.; Assenberg, R.; Ren, J.; Sainsbury, S.; Owens, R. J.; Stuart, D. I.; Grimes, J. M.; Harlos, K. Semi-automated microseeding of nanolitre crystallization experiments. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2008, 64, 14−18. (38) Owen, R. L.; Axford, D.; Nettleship, J. E.; Owens, R. J.; Robinson, J. I.; Morgan, A. W.; Dore, A. S.; Lebon, G.; Tate, C. G.; Fry, E. E.; Ren, J.; Stuart, D. I.; Evans, G. Outrunning free radicals in room-temperature macromolecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 810−818. (39) Owen, R. L.; Paterson, N.; Axford, D.; Aishima, J.; SchulzeBriese, C.; Ren, J.; Fry, E. E.; Stuart, D. I.; Evans, G. Exploiting fast detectors to enter a new dimension in room-temperature crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2014, 70, 1248−1256. (40) Winter, G.; Lobley, C. M.; Prince, S. M. Decision making in xia2. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1260−1273. (41) Waterman, D. G.; Winter, G.; Gildea, R. J.; Parkhurst, J. M.; Brewster, A. S.; Sauter, N. K.; Evans, G. Diffraction-geometry refinement in the DIALS framework. Acta Crystallogr. D Struct Biol. 2016, 72, 558−575. (42) Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367. (43) Janowski, P. A.; Moriarty, N. W.; Kelley, B. P.; Case, D. A.; York, D. M.; Adams, P. D.; Warren, G. L. Improved ligand geometries in crystallographic refinement using AFITT in PHENIX. Acta Crystallogr. D Struct Biol. 2016, 72, 1062−1072.

(44) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (45) 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. 1979, 134, 109−142. (46) Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2007, 2, 2728−2733. (47) DeLano, W. L.; Lam, J. W.PyMOL: A Communications Tool for Computational Models Abstracts of Papers of the American Chemical Society, 2005; 230, U1371U1372. (48) Laskowski, R. A.; Swindells, M. B. LigPlot+: multiple ligandprotein interaction diagrams for drug discovery. J. Chem. Inf. Model. 2011, 51, 2778−2786.

4945


Structures of Ebola Virus Glycoprotein Complexes with Tricyclic

Recommend Documents