Structures of Ebolavirus Glycoprotein Complexes with Tricyclic

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Structures of Ebolavirus Glycoprotein Complexes with Tricyclic Antidepressant and Antipsychotic Drugs Yuguang Zhao, Jingshan Ren, Elizabeth Evelyn Fry, Julia Xiao, Alain R Townsend, and David I. Stuart J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00350 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Journal of Medicinal Chemistry

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

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.

ABSTRACT A large number of Food and Drug Administration (FDA) approved drugs have been found to inhibit 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 the 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. 1 ACS Paragon Plus Environment

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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 the intracellular receptor NPC1 leading to transfer to the cytoplasm through membrane fusion.1, 911

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 pharmacological targets (Figure S1), all 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. RESULTS AND DISCUSSION Identification of GP−drug Interaction

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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 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 the fusion take 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 (Kds) derived from thermal−shift assay 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 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 molecule 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 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 VLPs.16 Table 1. Binding affinity to EBOV GP and inhibition activity. The standard deviation is shown in brackets. Toremifene is included for comparison. 3 ACS Paragon Plus Environment


∆Tm (°C)

Kd (µM)

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IC50 (µM) E-S-Flu VLP (MDCK-SIA1 cells)

eGFP-EBOV (vero cells)a

imipramine

-2

584 (256)

13.2 (0.3)

clomipramine

-3

118 (40)

10.3 (0.3)

11.4 (0.15)

7.8 (2.3)

6.24 (0.79)

0.15d

0.162 (0.048)

thioridazine toremifene

-15

16 (4)c

Ebola VLP (Hela cells)b 13.7 4.99

0.566

Values adapted, aJohansen et al.15; bKouznetsova et al.16; cZhao et al.24; dXiao et al.22. 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 datasets, extending to 2.35 Å resolution or better, were collected on beamlines of the Diamond 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 whilst 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−impramine, GP−clomipramine and GP−thioridazine have 376, 376 and 379 (out of 382) Cαs, matching with rmsds of 0.45 Å, 0.48 Å and 0.48 Å, respectively. Residues 46−52 preceding the disulphide 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


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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 disulphide 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 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 harbours 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 lid23, 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 Å 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 the largest toremifene 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 & 3A). 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 sub−pocket adjacent to α3. One benzene ring makes 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 towards 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 makes 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 make no strong protein interactions (Figures 3, S4). 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 stabilising the hydrophobic residues around it. Among all nine complexes we have now determined (including the three new structures described here), 6 ACS Paragon Plus Environment

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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 remainder23,24. 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 towards β1−β2 hairpin, presumably due to the great 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 towards molecule B of imipramine such that the chlorine group is positioned in front of Y517, increasing the overlap with the FF volume compared to molecule A of imipramine (Figure 4). The chlorine atom does not make any contact to the protein atoms, the benzene ring makes no contact with I185 and L515, and fewer interactions with I38 and L43 compared with the corresponding benzene ring of molecule A of imipramine, but clomipramine has better Kd and IC50, demonstrating that occupying the FF volume is pivotal for inhibitor binding (Figures 4 and S4). The Unique Binding Mode of Thioridazine Thioridazine has a dibenzthiazine tricyclic ring instead of the dibenzazepine in imipramine and clomipramine. Intriguingly thioridazine binds in the cavity in a dramatically different mode (Figure 5). Its dibenzthiazine is rotated ~110° from the dibenzazepine of clomipramine, 7 ACS Paragon Plus Environment


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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 2ethyl-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 making extensive interactions with side chains of I38, L43 and L186, as well as the main−chain atoms of V37 and L186; the other benzene ring contacts the side chains of M548 and I555, and the carbonyl oxygen of H549 (Figure 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. 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 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 make hydrogen bond with the side chain of D522. Toremifene is the most potent inhibitor characterized to date, with an IC50 50-fold better than clomipramine and


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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 the ring A of toremifene that binds in a subpocket adjacent to V66 and A101 with negatively charged electrostatic surface at the bottom would be likely to be more optimal for binding (Figures 6 and S1). Perhaps a chimeric molecule designed from the wellfitted fragments of several inhibitors might substantially improve potency beyond those identified to date. CONCLUSIONS The number of FDA approved drugs that have been found to inhibit Ebolavirus 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 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);

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 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



binding of the receptor NPC1. Protein residues lining the drug−binding cavity are highly conserved amongst the five known species of EBOVs, we therefore expect the nine drugs to bind the GP 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. EXPERIMENTAL SECTION Protein Expression and Purification The construct of EBOV (Zaire strain Mayinga−76) glycoprotein extracellular domain as

described earlier24 with one extra mutation H613A was cloned in the mammalian expression vector pNeosec.36 The resulting plasmid pNeosec−GP∆ has the mucin domain deleted and is tagged with a foldon trimerization sequence from the bacteriophage T4 fibritin and 6 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, UK). The transfection mixture was further supplemented with the mannosidase inhibitor kifunensine (Cayman chemical, Michigan, USA) at the final concentration of 5 µM to limit the protein glycosylation and facilitate crystallization. The conditioned media was collected 5 days after transfection and dialysed against PBS. The His−tagged protein was captured with talon beads (Takara Bio Europe SAS, France) at 15 for 1 hour 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, UK) and a buffer of 10 mM MES, pH 5.2, 150 mM NaCl.

Reagents 10 ACS Paragon Plus Environment

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Imipramine hydrochloride (Sigma−I0899) with specified purity of ≥ 99% and clomipramine hydrochloride (Sigma−C7291) with specified purity of ≥ 98% were purchased from SigmaAldrich. 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, 150 mM NaCl. Then the compounds were subjected to 2−fold serial dilution to the desired concentration with 10% DMSO of the above buffer. 25 µL each of diluted compounds in a semi−skired 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 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 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 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 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 pH5.2 and 100 nL microseed solution that was prepared by crushing a crystal about 50 × 50 × 50 µm3 in size in 50 µL reservoir solution by vortexing with a Teflon bead and then further diluted 50 times 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 on solubility. Eight crystals were soaked for each inhibitor in the above solutions for different lengths of time, ranging from 2 to 20 minutes.

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 frames of 0.1° rotation using synchrotron


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X-rays and PILATUS 6M detectors at Diamond Light Source, UK. GP-imipramine and GPclomipramine 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 s 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).38,39 360° of data were collected from every crystal that diffracted. 8 out of 8 crystals soaked with imipramine or thioridazine, and 3 out of 8 crystals soaked with clomipramine diffracted.

Data Processing, Structure Determination and Refinement Diffraction images were indexed, integrated and scaled with the automated data processing program Xia2 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 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, whilst GP−clomipramine and GP−thioridazine complexes use two crystals each. The resolution of the diffraction data for the three complexes ranges from 2.23 Å to 2.31 Å. Structure refinement used REFMAC542 or PHENIX43 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 omit electron density maps were calculated with CNS,46 volumes of the drug−binding cavity and drug molecules were



calculated with VOLUMES (Robert Esnouf, unpublished), figures were prepared with PyMOL47 and LigPlot.48

Supporting Information SMILES (CSV) Figures S1-S4 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. Accession Codes The coordinates and structure factors have been deposited with the RCSB Protein Data Bank under accession codes 6G9B, 6G9I and 6G95 for the 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 Yuguang Zhao: https://orcid.org/0000-0001-8916-8552 Jingshan Ren: https://orcid.org/0000-0003-4015-1404 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. analysed the results together


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with E.E.F., 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 interests.

Acknowledgments The authors would like to thank Diamond Light Source for beamtime (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 UK Medical Research Council (MR/N00065X/1). This is a contribution from the UK 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, Ebolavirus; FDA, Food and Drug Administration; GP, glycoprotein; VLP, virus-like particle.

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Figures

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,N-dimethylpropan-1-amine. (C) Thioridazine, 10[2-(1-methylpiperidin-2-yl)ethyl]-2-methylsulfanylphenothiazine.


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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 in red, and the glycan cap 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 surface representation. Structures of GP−toremifene (PDB ID, 5JQ7), GP−bepridil (PDB ID, 6F5U), GP−clomipramine (PDB ID, 6G9I) are first overlapped with GP−thioridazine (PDB ID, 6G95), 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.



Figure 3. Binding of imipramine. (A) Two imipramine molecules (grey sticks) are bound in the binding pocket (PDB ID, 6G9B). Protein main chains are shown as ribbons and side chains as sticks. The colour scheme is as in Figure 1. Side chains with large conformational changes in the apo GP structure are drawn as thinner grey sticks. (B) Comparison of the positions and orientations of the imipramines (grey 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.


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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 as sticks. The colour scheme is as in Figure 1. Side chains with large conformational changes in the apo GP (PDB ID, 5JQ3) structure are drawn as thinner grey sticks. (B) Comparison of the binding modes of clomipramine and imipramine (PDB ID, 6G9B), protein main chains and side chains are those associated with clomipramine.



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 as sticks. The colour scheme is as in Figure 1. Side chains with large conformational changes in the apo GP structure are drawn as thinner grey 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 shown in atom colours, side chains of the GP-clomipramine complex are shown as thinner grey sticks.


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



Table of Contents graphic


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Structures of Ebolavirus Glycoprotein Complexes with Tricyclic

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