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. 2020 Jan 3;21(1):334.
doi: 10.3390/ijms21010334.

GABAA Receptor Ligands Often Interact with Binding Sites in the Transmembrane Domain and in the Extracellular Domain-Can the Promiscuity Code Be Cracked?

Maria Teresa Iorio  1 , Florian Daniel Vogel  2 , Filip Koniuszewski  2 , Petra Scholze  3 , Sabah Rehman  2 , Xenia Simeone  2 , Michael Schnürch  1 , Marko D Mihovilovic  1 , Margot Ernst  2
Affiliations

GABAA Receptor Ligands Often Interact with Binding Sites in the Transmembrane Domain and in the Extracellular Domain-Can the Promiscuity Code Be Cracked?

Maria Teresa Iorio et al. Int J Mol Sci. .

Abstract

Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular "canonical" site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/β- sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.

Keywords: GABAA; benzodiazepines (BZ); binding sites; etomidate; receptors.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Scheme A1
Scheme A1
Reagents and conditions: (a) ethylpyruvate, AcOH, EtOH, rf, 2 h, 95%; (b) PPA, 110 °C, 5 h, 60%; (c) DMF, K2CO3, 0 °C, 30 min, 70%; (d) NaOH (2N), rf, 3 h, 60%.
Scheme A2
Scheme A2
Reagents and conditions (a) diethyl(ethoxymethylene)malonate (1 equiv.), toluene, 110 °C, 24 h; (b) diphenylether, 235 °C, 1 h; (c) phosphoryl chloride (1 mL/mmol); 2 h, (d) p-bromo phenylhydrazine (1.1 equiv.), triethylamine (2.2 equiv.), ethanol, 24 h.
Figure A1
Figure A1
Gaussian calculation of two configurations of MTI163. The structure forming a 7-membered ring via an intramolecular hydrogen bond (a) is approximately 11 kcal/mol more stable than a conformation without such an intramolecular H-bond (b).
Figure A2
Figure A2
Diversity of etomidate binding poses in 6HUP. Of the top 100 poses (GoldScore ranking), consensus scoring and ligand flexibility suggests that the diversity of the posing space is very high and experimentally guided constraints or a higher level of computational theory is needed. The diversity of the posing space is illustrated based on the most diverse solutions among the top 20 from two scoring functions. All poses display a high overlap of ligand–protein interactions with the diazepam present in this site in 6HUP. (a) Front view; (b) side view.
Figure A3
Figure A3
Different binding poses of MTI163 in 6HUP. Top 20 poses from GoldScore and ChemScore ranking were analysed, and the seven poses thatremain among top 20 by consensus (i.e., in ranks 1–20 in both scoring functions) are depicted here. (a) Front view; (b) Side view.
Figure A4
Figure A4
Different binding poses of MTI163 in the N265S mutant. There is no ligand bound structure with the β1 subunit, thus 6HUP was point mutated for the docking. In this case, five docking poses were found in the top 20 of GoldScore and ChemScore. (a) Front view; (b) Side view; (c) Comparison between the pose displayed in Figure 5 and the most similar pose observed in the N265S mutant. The two bound complexes are superposed (reflected by the two-colored ribbon rendering), the 6HUP (wild type β3) pose and N265 are rendered as light green sticks, the S265 and the corresponding MTI163 pose are rendered as yellow sticks.
Figure A5
Figure A5
Concentration-dependent MTI163 modulation of GABA EC3–5 elicited current in α1β1. Response at each point of measurement is represented as individual data point as Mean ±  SEM (n = 4, EC50 = 7.65 µM, nH = 0.78).
Figure 1
Figure 1
(a) Schematic rendering of a GABAA receptor. The extracellular domain (ECD) and transmembrane domain (TMD) contain various binding sites [11]. The binding sites studied in this work are at interfaces formed by a principal (+) subunit face and a complementary (−) subunit face. The shape resembles vaguely a space filling rendering of the protein’s ECD and TMD, while intracellular domain (ICD) shape is a purely schematic rendering based on more remote homologues. (b) Schematic planes through the ECD (top) and TMD (bottom) of a canonical αβγ receptor, the GABA sites are indicated as dark grey ellipses. Site 1 (blue), the high affinity benzodiazepine site, is at the ECD– α+/γ− interface; site 2 (blue), which confers modulatory effects of pyrazoloquinolinone ligands is at the ECD– α+/β− interface, and site 3 (orange), the etomidate site [7] occurs twice and is located at the TMD– β+/α− interfaces below the GABA binding sites.
Figure 2
Figure 2
(a) Structure of MTI163; (b) structure of DCBS192; (c) pharmacophores of indole derivatives (left) and of PQs (right); (d) superposition of pharmacophores of PQ and indole derivatives. Panels c and d depict the pharmacophores already discussed in [12].
Figure 3
Figure 3
Radioligand displacement assay of [3H]-Flunitrazepam by DCBS192 (a) and MTI163 (b). Membranes from rat cerebellum were incubated with 2 nM [3H]-flunitrazepam in the presence of various concentrations of the two ligands. Data shown are the mean ± SEM of three independent experiments performed in duplicates each. (c,d) Superposition of the flumazenil bound 6D6T (green) and the alprazolam bound 6HUO (yellow) in two different perspectives demonstrate that the structurally related molecules do not bind with aligned pharmacophore features at all. The heteroatoms of the molecules are depicted in different colors: blue-nitrogen, red-oxygen, green-chloro. (e) Structures of alprazolam and flumazenil.
Figure 4
Figure 4
(a) Concentration-dependent MTI163 modulation of GABA EC3-5 elicited current in α1β1, α1β2, α1β3 and α1β3γ2cct. Response at each point of measurement is represented as mean ± SEM (n = 3–4). Response at 10 µM is highlighted in red color. The y-axis is broken at indicated values for better visualization of all curves in the panel. For better visualization, a full scale version of the α1β1 dose response curve is in the Appendix A (Figure A5). (b) Comparison of MTI163 modulation of GABA3-5 elicited current in α1β1, α1β2, α1β2N265S, α1β3, and α1β3N41R receptors at 10 µM. Bars are given as mean ± SEM (n = 4–8). Each individual data point is displayed by a dot. Statistically significant differences were determined by one-way ANOVA with Tukey’s multiple comparisons post-hoc test, *** corresponds to p < 0.0001.
Figure 5
Figure 5
Representative docking pose of MTI163 in the diazepam pocket of 6HUP. (a) The MTI163 pose, which is fully consistent with all experimental evidence, is displayed with the pocket-forming amino acids that were set flexible during docking. (b) Overlay of diazepam (yellow) and the MTI163 (red) pose in 6HUP ligands structures (top), ligands surfaces (bottom). The left subunit (pink) is β3, the right one (blue) is α1.
Figure 6
Figure 6
Graphical overview of chemical scaffolds which bind to distinctive interfaces at αβγ GABAA receptors. The three binding sites are depicted as colored ellipses: site 1, ECD- α+/γ−: blue; site 2, ECD- α+/β−: orange; site 3, TMD- β+/α− red. Substances that bind at more than one site are placed at the intersection of the different binding sites. All placements rest on current evidence from structural studies [4,17] and decades of mutational studies [19,20,21,23,24,25,26]. Some of the placements are thus tentative. Of note, for both the benzodiazepine site and the “etomidate site”, many more ligands have been described, partly reviewed by Sieghart (2015) and Sieghart and Savic (2018) [1,10]; however, low affinity interactions with other sites have not been regularly investigated.
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