Target Identification and Mode of Action of Four Chemically Divergent Drugs against Ebolavirus Infection

Here, we show that four chemically divergent approved drugs reported to inhibit Ebolavirus infection, benztropine, bepridil, paroxetine and sertraline, directly interact with the Ebolavirus glycoprotein. Binding of these drugs destabilizes the protein, suggesting that this may be the mechanism of inhibition, as reported for the anticancer drug toremifene and the painkiller ibuprofen, which bind in the same large cavity on the glycoprotein. Crystal structures show that the position of binding and the mode of interaction within the pocket vary significantly between these compounds. The binding constants (<i>K</i>_d) determined by thermal shift assay correlate with the protein–inhibitor interactions as well as with the antiviral activities determined by virus cell entry assays, supporting the hypothesis that these drugs inhibit viral entry by binding the glycoprotein and destabilizing the prefusion conformation. Details of the protein–inhibitor interactions of these complexes and their relation with binding affinity may facilitate the design of more potent inhibitors

Structures of Ebola Virus Glycoprotein Complexes with Tricyclic Antidepressant and Antipsychotic Drugs

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

Structures of Ebola Virus Glycoprotein Complexes with Tricyclic Antidepressant and Antipsychotic Drugs

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

Stabilising interactions at the inter-pentameric interfaces of the inside-out particle.

<p>Interface analysis of the inside-out particle. (A) Black rectangles high-light the interacting regions between the 2- and 3-fold icosahedral symmetry axes and around the 3-fold symmetry axis with the proteins drawn as ribbons coloured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006607#ppat.1006607.g003" target="_blank">Fig 3</a>. (B) Blue mesh electron density with the VP2 (green) and VP3 (red) loops shown interacting at the two-fold axis with residues involved labelled. (C) The symmetry-related copies of VP2 and VP3 from three pentamers in close proximity at the three-fold axis, The VP2 residues might also form capsid-stabilising interactions.</p

Conformational changes highlighting the differences between the native FMDV capsid and the inside-out particle.

<p>Depth-cued structures, after VP1 superposition, of native (yellow cartoon) and inside-out particle protomers (purple cartoon), highlighting the conformational changes in VP3 (red circles). (A) The shift in VP3 beta-sheets in the inside-out particle is highlighted by red circles. (B) The 20° y-axis rotated view of the protomers emphasises the 4.6° rotational shift in VP3 (red arrow). (D) The structural clashes which would occur between the purple protomers at the VP2-VP3 two-fold axis, had they assembled in the same orientation as the native, are highlighted by the black circles, and a (C) native arrangement of protomers is shown for comparison.</p

5.2 Å structure of the FMDV inside-out particle.

<p>(A and B) show the particle and a cross-section through the particle viewed down the two-fold icosahedral symmetry axis, coloured radially from the centre (<110 Å: red; 120–140 Å: yellow; >150 Å: blue). (C) Atomic fitting of the structure into the electron density map (transparent grey render) to generate the whole virus structure of the inside-out particle. Individual protein chains are coloured blue: VP1, green: VP2 and red: VP3.</p

Atomic model fitting of the inside-out particle into the EM density.

<p>The 5.2 Å electron density map (blue mesh) of the inside-out particle allowed the unambiguous fitting and refinement of the PDB (1ZBE) model. Stereo-views of the same region with the (A) C-alpha backbone and the (B) atomic representation of the model fitted into the EM density are shown in red.</p

Analysis of the inside-out FMDV particles.

<p>(A) Sucrose gradient analysis of particles before and after dissociation to pentamers. (B) Negative stain EM analysis of dissociated particles before concentration and (C) after 40-fold concentration. (D) Cryo-electron microscopy images of low-pH treated FMDV capsids. Red square: empty inside-out particles with stain inside. Red circle: pentamer, side view. Yellow circle: pentamer, top view and Red asterisk: a small proportion of residual native particles.</p

A comparison of the native FMDV capsid structure to that of the inside-out particle.

<p>A comparison of surface representations of the (A) native crystal structure (1ZBE) and the (B) inside-out particle viewed down the 2-fold icosahedral symmetry axis. A protomer comprising of VP1 (blue), VP2 (green) and VP3 (red) is highlighted by a black triangle. A gap at the two-fold axis, and the reverse orientation of VP2 and VP3 can be seen in the inside-out particle.</p

Atomic model fitting of the inside-out particle pentamer structure into the isolated pentamer density.

<p>The isolated pentamer density (grey mesh) is observed down the icosahedral 5-fold axis from the (A) inside and (B) outside (relative to the inside-out pentamer) showing how the VP1, VP2 and VP3 (cartoon representation coloured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006607#ppat.1006607.g003" target="_blank">Fig 3</a>) are arranged in a dissociated pentamer. (C) A zoomed-in view to show the quality of the fitting.</p