[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]
Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site
Jiri Wald, Marion Pasin, Martina Richter, Christin Walther, Neann Mathai, Johannes Kirchmair, Vadim A. Makarov, Nikolaus Goessweiner-Mohr, Thomas C. Marlovits, Irene Zanella, Antonio Real-Hohn, Nuria Verdaguer, Dieter Blaas, and Michaela Schmidtke
PNAS first published August 28, 2019 / DOI: https://doi.org/10.1073/pnas.1904732116
Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved August 1, 2019 (received for review March 29, 2019)
Significance
More than 160 rhinovirus (RV) types cause about a billion respiratory infections annually in the United States alone, contributing to influenza-like illness. This diversity makes vaccination impractical. Existing small-molecule inhibitors target RVs by binding to a hydrophobic pocket in the capsid but exhibit side effects, resistance, and/or mutational escape, impeding registration as drugs. The pyrazolopyrimidine OBR-5-340 acts like other capsid binders by preventing conformational changes required for genome release. However, by using cryo-EM, we show that OBR-5-340 inhibits the naturally pleconaril-resistant RV-B5 by attaching close to the pocket entrance in a binding geometry different from that of most capsid binders. Combinations of inhibitors with disparate binding modes might thus effectively combat RVs while reducing the risk of resistance development.
Abstract
Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with less-conserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors.
rhinovirus – capsid binder – inhibitor – 3D structure – cryo-EM
Footnotes
1 J.W., M.P., M.R., and C.W. contributed equally to this work.
2 To whom correspondence may be addressed. Email: dieter.blaas@meduniwien.ac.at or michaela.schmidtke@med.uni-jena.de.
Author contributions: J.K., T.C.M., A.R-H., N.V., D.B., and M.S. designed research; J.K, T.C.M, D.B., and M.S. supervised research; J.W., M.P., M.R., C.W., N.M., J.K., I.Z., A.R-H., and D.B. performed research; V.A.M. contributed new reagents/analytic tools; J.W., M.P., M.R., C.W., N.M., J.K., N.G-M., A.R-H., D.B., and M.S. analyzed data; D.B. reconstructed and refined the cryo-EM data; M.P. reconstructed and refined the cryo-EM data and carried out the thermal stability assays; M.R., C.W., and M.S. carried out the antiviral tests; N.M. analyzed binding sites of all compounds; I.Z. carried out stability assays; N.V. critically assessed the manuscript; and N.M., J.K., D.B., and M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The 3D structures reported in this paper have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, http://www.rcsb.org/ and the Electron Microscopy Data Bank, https://www.ebi.ac.uk/pdbe/emdb/ (accession nos.: RV-B5 complexed to OBR-5-340, PDB 6SK5 and EMD-10220; RV-B5, PDB 6SK6 and EMD-10221; RV-A89, PDB 6SK7and EMD-10222).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904732116/-/DCSupplemental.
Published under the PNAS license.
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Keywords: Antivirals; Drugs Resistance; Pleconaril; Enterovirus; Rhinovirus.
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