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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Sieghart, W. Structure and pharmacology of γ-aminobutyric acid A receptor subtypes. Pharmacol. Rev. 47, 181–234 (1995).

    CAS  PubMed  Google Scholar 

  2. 2

    Sigel, E. Mapping of the benzodiazepine recognition site on GABAA receptors. Curr. Top. Med. Chem. 2, 833–839 (2002).

    CAS  Article  Google Scholar 

  3. 3

    Miller, P.S. & Smart, T.G. Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol. Sci. 31, 161–174 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Clayton, T. et al. An updated unified pharmacophore model of the benzodiazepine binding site on γ-aminobutyric acidA receptors: correlation with comparative models. Curr. Med. Chem. 14, 2755–2775 (2007).

    CAS  Article  Google Scholar 

  5. 5

    Mokrab, Y. et al. Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor. J. Mol. Graph. Model. 26, 760–774 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Ci, S., Ren, T. & Su, Z. Investigating the putative binding-mode of GABA and diazepam within GABAA receptor using molecular modeling. Protein J. 27, 71–78 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Berezhnoy, D., Gibbs, T.T. & Farb, D.H. Docking of 1,4-benzodiazepines in the α1/γ2 GABAA receptor modulator site. Mol. Pharmacol. 76, 440–450 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Sancar, F., Ericksen, S.S., Kucken, A.M., Teissére, J.A. & Czajkowski, C. Structural determinants for high-affinity zolpidem binding to GABAA receptors. Mol. Pharmacol. 71, 38–46 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Evers, A., Gohlke, H. & Klebe, G. Ligand-supported homology modelling of protein binding-sites using knowledge-based potentials. J. Mol. Biol. 334, 327–345 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Rarey, M., Kramer, B., Lengauer, T. & Klebe, G. A fast flexible docking method using an incremental construction algorithm. J. Mol. Biol. 261, 470–489 (1996).

    CAS  Article  Google Scholar 

  12. 12

    Moitessier, N., Englebienne, P., Lee, D., Lawandi, J. & Corbeil, C.R. Towards the development of universal, fast and highly accurate docking/scoring methods: a long way to go. Br. J. Pharmacol. 153 (suppl 1): S7–S26 (2008).

    CAS  PubMed  Google Scholar 

  13. 13

    Claussen, H., Buning, C., Rarey, M. & Lengauer, T. FlexE: efficient molecular docking considering protein structure variations. J. Mol. Biol. 308, 377–395 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Chema, D., Eren, D., Yayon, A., Goldblum, A. & Zaliani, A. Identifying the binding mode of a molecular scaffold. J. Comput. Aided Mol. Des. 18, 23–40 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Wang, R., Lai, L. & Wang, S. Further development and validation of empirical scoring functions for structure-based binding affinity prediction. J. Comput. Aided Mol. Des. 16, 11–26 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Labute, P. The generalized Born/volume integral implicit solvent model: estimation of the free energy of hydration using London dispersion instead of atomic surface area. J. Comput. Chem. 29, 1693–1698 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Duncalfe, L.L., Carpenter, M.R., Smillie, L.B., Martin, I.L. & Dunn, S.M. The major site of photoaffinity labeling of the γ-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the α subunit. J. Biol. Chem. 271, 9209–9214 (1996).

    CAS  Article  Google Scholar 

  18. 18

    Berezhnoy, D. et al. On the benzodiazepine binding pocket in GABAA receptors. J. Biol. Chem. 279, 3160–3168 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Wieland, H.A., Lüddens, H. & Seeburg, P.H. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J. Biol. Chem. 267, 1426–1429 (1992).

    CAS  PubMed  Google Scholar 

  20. 20

    Davies, M., Bateson, A.N. & Dunn, S.M. Structural requirements for ligand interactions at the benzodiazepine recognition site of the GABAA receptor. J. Neurochem. 70, 2188–2194 (1998).

    CAS  Article  Google Scholar 

  21. 21

    Buhr, A., Schaerer, M.T., Baur, R. & Sigel, E. Residues at positions 206 and 209 of the α1 subunit of γ-aminobutyric acidA receptors influence affinities for benzodiazepine binding site ligands. Mol. Pharmacol. 52, 676–682 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Sawyer, G.W., Chiara, D.C., Olsen, R.W. & Cohen, J.B. Identification of the bovine γ-aminobutyric acid type A receptor α subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15–4513. J. Biol. Chem. 277, 50036–50045 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Tan, K.R. et al. Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of γ-aminobutyric acid type A receptors: superposition of different allosteric modulators. J. Biol. Chem. 282, 26316–26325 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Sigel, E., Schaerer, M.T., Buhr, A. & Baur, R. The benzodiazepine binding pocket of recombinant α1β2γ2 γ-aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains. Mol. Pharmacol. 54, 1097–1105 (1998).

    CAS  Article  Google Scholar 

  25. 25

    Buhr, A., Baur, R. & Sigel, E. Subtle changes in residue 77 of the γ subunit of α1β2γ2 GABAA receptors drastically alter the affinity for ligands of the benzodiazepine binding site. J. Biol. Chem. 272, 11799–11804 (1997).

    CAS  Article  Google Scholar 

  26. 26

    Amin, J., Brooks-Kayal, A. & Weiss, D.S. Two tyrosine residues on the α subunit are crucial for benzodiazepine binding and allosteric modulation of γ-aminobutyric acidA receptors. Mol. Pharmacol. 51, 833–841 (1997).

    CAS  Article  Google Scholar 

  27. 27

    Derry, J.M.C., Dunn, S.M.J. & Davies, M. Identification of a residue in the γ-aminobutyric acid type A receptor α subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding. J. Neurochem. 88, 1431–1438 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Tan, K.R., Baur, R., Charon, S., Goeldner, M. & Sigel, E. Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABAA receptors. J. Neurochem. 111, 1264–1273 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Kucken, A.M., Teissére, J.A., Seffinga-Clark, J., Wagner, D.A. & Czajkowski, C. Structural requirements for imidazobenzodiazepine binding to GABAA receptors. Mol. Pharmacol. 63, 289–296 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Huang, N., Shoichet, B.K. & Irwin, J.J. Benchmarking sets for molecular docking. J. Med. Chem. 49, 6789–6801 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Mysinger, M.M. & Shoichet, B.K. Rapid context-dependent ligand desolvation in molecular docking. J. Chem. Inf. Model. 50, 1561–1573 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Wolber, G. & Langer, T. LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters. J. Chem. Inf. Model. 45, 160–169 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Wei, B.Q., Baase, W.A., Weaver, L.H., Matthews, B.W. & Shoichet, B.K. A model binding site for testing scoring functions in molecular docking. J. Mol. Biol. 322, 339–355 (2002).

    CAS  Article  Google Scholar 

  34. 34

    Ramerstorfer, J. et al. The GABAA receptor α+β- interface: a novel target for subtype selective drugs. J. Neurosci. 31, 870–877 (2011).

    CAS  Article  Google Scholar 

  35. 35

    Popp, F.D., Parson, R. & Donigan, B.E. Synthesis of potential anticonvulsants: condensation of isatins with acetone and related ketones. J. Pharm. Sci. 69, 1235–1237 (1980).

    CAS  Article  Google Scholar 

  36. 36

    Marcou, G. & Rognan, D. Optimizing fragment and scaffold docking by use of molecular interaction fingerprints. J. Chem. Inf. Model. 47, 195–207 (2007).

    CAS  Article  Google Scholar 

  37. 37

    Korb, O., Stutzle, T. & Exner, T.E. Empirical scoring functions for advanced protein-ligand docking with PLANTS. J. Chem. Inf. Model. 49, 84–96 (2009).

    CAS  Article  Google Scholar 

  38. 38

    Mihic, S.J., Whiting, P.J., Klein, R.L., Wafford, K.A. & Harris, R.A. A single amino acid of the human γ-aminobutyric acid type A receptor γ2 subunit determines benzodiazepine efficacy. J. Biol. Chem. 269, 32768–32773 (1994).

    CAS  PubMed  Google Scholar 

  39. 39

    de Graaf, C. & Rognan, D. Selective structure-based virtual screening for full and partial agonists of the β2 adrenergic receptor. J. Med. Chem. 51, 4978–4985 (2008).

    CAS  Article  Google Scholar 

  40. 40

    Verheij, M.H. et al. Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H and the ion channel serotonin 5-HTA. Bioorg. Med. Chem. Lett. 21, 5460–5464 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Venhorst, J., Nunez, S., Terpstra, J.W. & Kruse, C.G. Assessment of scaffold hopping efficiency by use of molecular interaction fingerprints. J. Med. Chem. 51, 3222–3229 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Hibbs, R.E. & Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011).

    CAS  Article  Google Scholar 

  43. 43

    Michino, M. et al. Community-wide assessment of GPCR structure modelling and ligand docking: GPCR Dock 2008. Nat. Rev. Drug Discov. 8, 455–463 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  Article  Google Scholar 

  45. 45

    Shi, J., Blundell, T.L. & Mizuguchi, K. FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 310, 243–257 (2001).

    CAS  Article  Google Scholar 

  46. 46

    Sali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    CAS  Article  Google Scholar 

  47. 47

    Sieghart, W. & Schuster, A. Affinity of various ligands for benzodiazepine receptors in rat cerebellum and hippocampus. Biochem. Pharmacol. 33, 4033–4038 (1984).

    CAS  Article  Google Scholar 

  48. 48

    Zhang, W., Koehler, K.F., Zhang, P. & Cook, J.M. Development of a comprehensive pharmacophore model for the benzodiazepine receptor. Drug Des. Discov. 12, 193–248 (1995).

    CAS  PubMed  Google Scholar 

Download references


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.

Author information




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.

Corresponding author

Correspondence to Margot Ernst.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing