Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands

Abstract

Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABAA receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an unbiased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design.

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Figure 1: Structure of diazepam-sensitive GABAARs and of benzodiazepine ligands.
Figure 2: CBM candidates.
Figure 3: Representation of a CBM I pose of diazepam.
Figure 4: CBM I reference binding modes of diazepam and flumazenil compared to docking poses of virtual screening hits 19 and 20, selected by IFP scoring.
Figure 5: 3-hydroxyoxindoles as a new class of benzodiazepine-binding-site ligands of GABAARs.

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Acknowledgements

Financial support from the Doctoral Fellowship Programme of the Austrian Academy of Science (L.R.), the Austrian Science Fund grant P19653 (M.E.) and the European Commission Seventh Framework Programme grant HEALTH-F4-2008-202088 (W.S. and I.J.P.d.E.) is gratefully acknowledged. We also thank Inte:Ligand for providing us a free license for LigandScout 3.0 and Open Eye for an Omega license. We thank M. Mysinger and J. Irwin from the Shoichet laboratory for generating the focused 'benzodiazepine' decoy set. In addition we thank E. Sigel for helpful suggestions and discussions during generation of this work; M. Stojanovic for technical assistance in the binding assays; A. Chalikiopoulos and M. Verheij for assistance in fragment docking studies; and E. Urban, S. Haselmaier, J. König, H. Custers and A. van de Stolpe for providing HRMS and 1H- and 13C-NMR spectral data.

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L.R., W.S., G.F.E., I.J.P.d.E., C.d.G. and M.E. contributed to the design and evaluation of experiments. Docking and virtual screening experiments were performed by L.R. (supervised by G.F.E.) and C.d.G. GABAAR models were generated and evaluated by M.E. and M.M. Radioligand and electrophysiological experiments were performed by Z.V. The manuscript was written by W.S., L.R., G.F.E., I.J.P.d.E., C.d.G. and M.E., and all authors commented on and helped to revise the text.

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Correspondence to Margot Ernst.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 7474 kb)

Supplementary Model 1

CBM I bound complex of compound 1 (PDB 549 kb)

Supplementary Model 11

CBM I bound complex of compound 11 (PDB 549 kb)

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Richter, L., de Graaf, C., Sieghart, W. et al. Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands. Nat Chem Biol 8, 455–464 (2012). https://doi.org/10.1038/nchembio.917

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