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More-powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules

Nature Structural & Molecular Biology volume 21, pages 282–288 (2014)Cite this article

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Abstract

Enterovirus 71 (HEV71) epidemics in children and infants result mainly in mild symptoms; however, especially in the Asia-Pacific region, infection can be fatal. At present, no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics–enhanced ligand docking and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (with IC50 of 25 pM) is an order of magnitude more potent than the best previously reported inhibitor and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.

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Figure 1: The inhibitor-binding site and selected inhibitor structures.
Figure 2: Real-space averaged |Fo| − |Fc| omit maps (green mesh) of four 3-(4-pyridyl)-2-imidazolidinone derivatives bound to HEV71.
Figure 3: GPP3 bound to VP1 and thermal stabilization by GPP2, GPP3 and GPV13.
Figure 4: VP1 pocket and docking of the new ligands.
Figure 5: Characterization of newly designed capsid binders bound to the VP1 pocket of HEV71.

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Acknowledgements

We thank A. Kotecha for assistance with Diamond data collection and the beamline staff at Diamond light source beamlines I03 and I24 for expert assistance and advice. GPP and GPV compounds were made by F. Prauchart (Department of Pharmaceutical Chemistry, University of Innsbruck). MS analyses were carried out by C. Schofield, and P. Abrusci helped with the Sigma Plot program. Administrative and high-performance computing was supported by the Wellcome Trust Core Award, grant no. 090532/Z, and particular help was provided by R. Esnouf. Work was supported by the Chinese National Major Project of Infectious Disease, Ministry of Science and Technology 973 Project (grant nos. 2011CB910300 and 2014CB542800) and Major National Science and Technology Programs (grant no. 2012ZX10004701). D.I.S., E.E.F. and T.S.W. are supported by the UK Medical Research Council (G110525 and G100099), J.R. by the Wellcome Trust, J.K. by Sanofi Pasteur and L.D.C. by the World Health Organization. Research leading to these results received funding from the European Union FP7, SILVER grant no. 260644.

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Author notes

  1. Luigi De Colibus and Xiangxi Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Structural Biology, University of Oxford, Oxford, UK

    Luigi De Colibus, John A B Spyrou, Jingshan Ren, Jonathan Grimes, Thomas S Walter, Elizabeth E Fry & David I Stuart

  2. National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, China

    Xiangxi Wang, Xuemei Li, Wei Peng & Zihe Rao

  3. School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK

    James Kelly, Nicola Stonehouse & David J Rowlands

  4. Diamond Light Source, Didcot, UK

    Jonathan Grimes & David I Stuart

  5. Department of Pharmaceutical Chemistry, University of Innsbruck, Innsbruck, Austria

    Gerhard Puerstinger

  6. National Institutes for Food and Drug Control, No. 2, Tiantan Xili, Beijing, China

    Zhongyu Hu & Junzhi Wang

  7. Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing, China

    Zihe Rao

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Contributions

D.I.S. and Z.R. supervised and coordinated the project; J.W., Z.H., X.L., G.P. and W.P. made samples available; X.W. purified and crystallized the samples and performed thermofluor experiments; G.P. and J.G. provided the GPP ligands; L.D.C. designed ALD and NLD; L.D.C. and J.A.B.S. ran the in silico docking and analyzed data under supervision by D.I.S.; L.D.C. and T.S.W. soaked crystals for data collection, which was performed by L.D.C., J.A.B.S., J.R. and E.E.F.; L.D.C., J.A.B.S., J.R. and D.I.S. contributed to data processing, structure determination and model building; J.K. performed the in vitro TCID50 assay and together with N.S. and D.J.R. analyzed the data. L.D.C., E.E.F. and D.I.S., in discussion with J.R., D.J.R. and Z.R., wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Zihe Rao or David I Stuart.

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Integrated supplementary information

Supplementary Figure 1 Comparison of complexes of EV71 with sphingosine and GPP2.

Sphingosine (blue) - EV71 VP1 (blue) (PDBID: 3VBF), superimposed on GPP2 (magenta) - EV71 VP1 (cyan). The RMS difference between all Cα atoms of the icosahedral protomer is 0.4 Å.

Supplementary Figure 2 Predicted binding affinity (GOLD score or ΔG) versus experimental –log(IC50 (pIC50) values.

The compounds are the 3-(4-Pyridyl)-2-imidazolidinone derivatives listed in Supplementary Table 1. The calculated points for ALD and NLD are in red whilst the experimental values are reported in yellow on the plot. Plot (a) shows results for GOLD (with GOLD fitness scores), (b) for Glide (energy of interaction expressed as van der Waals plus electrostatic interactions) (c) for Rosetta (interface_delta), (d) GOLD/AMBER (ΔG of binding). For details of the protocols see the methods section of the main text. The ligand 42-8e reported in the Supplementary Table 1 could not be docked into the crystal structure using Glide. The following ligands: 2-d-21, 10-26, 4-24 reported in the Supplementary Table 1 were excluded from the Rosetta plot. They represent outlier points, because these molecules were partially docked in the binding.

Supplementary Figure 3 Stereo diagrams comparing the results of the various docking protocols for GPP2.

The side chain of Ile113 is not shown, to reveal the hydrogen bond, drawn as dotted lines, between the main chain nitrogen of this residue and the ligand oxygen. Residues from VP1 are shown in blue and Ile24 from VP3 in orange. (a) Overlay of the docked ligand GPP2 (light green) by QMPLD method and the structurally determined conformation of GPP2 (magenta) (RMSD for all inhibitor atoms 1.3 Å). (b) Overlay of the docked ligand GPP2 by GOLD (violet) and the structurally determined conformation of GPP2 (magenta) (RMSD for all inhibitor atoms 0.9 Å). (c) Overlay of the docked ligand GPP2 by Rosetta (gray) and the structurally determined conformation of GPP2 (magenta) (note GPP2 is docked upside down).

Supplementary Figure 4 Docking of GPP3 and NLD molecules in HEV71 crystal structures.

(a) Docking of GPP3 by QMPLD into the 2.7 Å crystal structure reported by Plevka et al. 2013 (reference 15 in main text, PDBID:3ZFE). In cyan the sphingosine molecule found in the 3ZFE crystal structure and in grey GPP3 docked by QMPLD. The dashed line represents the hydrogen bond between GPP3 and Ile113 of VP1. (b) In magenta the GPP3 molecule found in the crystal structure and in grey GPP3 molecule docked by QMPLD. The dashed line represents the hydrogen bond between GPP3 and Ile113 of VP1. The residue Phe135 is at the front whereas residue Phe155 is towards the back of the figure. Residue Ile24 of VP3 is coloured orange. The RMSD between the two virus structures (all atoms) is 0.8 Å, whilst the RMSD between the two GPP3 positions is 1.6 Å. (c) Docking of GPP3 by QMPLD into the 3.7 Å crystal structure reported by Plevka et al. 2013 (reference 14 in main text, PDBID:4AED) with the experimentally observed conformation. In gray GPP3 molecule docked by QMPLD into the 4AED crystal structure and in magenta the experimentally observed conformation of GPP3 (obtained by superposition of our complex onto the 4AED structure). (d) In grey the protonated form of the GPP3 molecule docked by QMPLD into the 4AED crystal structure and in magenta the experimentally observed conformation of GPP3. The dashed line represents the hydrogen bond established by GPP3 and residue Ile113 of VP1. Residue Phe135 is at the front whereas residue Phe155 is towards the back of the figure. The RMSD between the two virus structures (4AED and our complex, all atoms) is 1.1 Å, whilst the RMSD between the two GPP3 positions shown in (c) is 13.3 Å (GPP3 is docked upside down) and that between the two GPP3 positions in shown in (d) is 1.8 Å. (e) Docking of NLD by QMPLD into the 2.7 Å crystal structure reported by Plevka et al. 2013 (reference 15 in main text, PDBID:3ZFE) with the experimentally observed conformation. In cyan the sphingosine molecule found in the 3ZFE crystal structure and in grey NLD molecule docked by QMPLD. The dashed line represents the hydrogen bond established by GPP3 and residue Ile113 of VP1. (f) In magenta the NLD molecule found in the crystal structure and in grey NLD docked by QMPLD. The dashed line is the hydrogen bond between NLD and Ile113 and Gln202 of VP1. Phe135 is towards the front and Phe155 towards the back of the figure. Residue Ile24 of VP3 is coloured orange. The RMSD between the two virus structures (all atoms) is 0.8 Å, whilst the RMSD between the two NLD positions is 1.6 Å.

Supplementary Figure 5 Mass spectrum of ALD and NLD compounds.

(a) Upper panel, experimental peaks with the associated molecular mass values. The main species in the sample has a measured molecular mass of 476.2266 m/z corresponding to the introduction of an amide group on the pyridine moiety of 3-(4-pyridyl)-2-imidazolidinone. The lower panel shows the theoretical molecular mass for this compound. (b) Upper panel shows the experimental peaks with the associated molecular mass values. The main specie in the sample has a measured molecular mass of 448.2311 m/z corresponding to the introduction of an amine group on the pyridine moiety of 3-(4-pyridyl)-2-imidazolidinone. The lower panel shows the theoretical molecular mass for this compound.

Supplementary Figure 6 Structural comparison of key residues involved in forming a hydrophobic trap for pocket binders.

Structurally equivalent residues within VP1 pocket of EV71 (blue), Poliovirus type 2 (yellow, PDBID:1EAH) and rhinovirus 14 (orange, PDBID:1NA1). These residues are critical for positioning the compounds within the pocket.

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De Colibus, L., Wang, X., Spyrou, J. et al. More-powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules. Nat Struct Mol Biol 21, 282–288 (2014). https://doi.org/10.1038/nsmb.2769

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