International Journal of Molecular Sciences Article 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 and Margot Ernst 2,* 1 Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163-OC, 1060 Vienna, Austria; [email protected] (M.T.I.); [email protected] (M.S.); [email protected] (M.D.M.) 2 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090 Vienna, Austria; fl[email protected] (F.D.V.); fi[email protected] (F.K.); [email protected] (S.R.); [email protected] (X.S.) 3 Department of Pathobiology of the Nervous System, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090 Vienna, Austria; [email protected] * Correspondence: [email protected]; Tel.: +43-1-40160-34065 Received: 29 November 2019; Accepted: 26 December 2019; Published: 3 January 2020 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: receptors; GABAA; benzodiazepines (BZ); binding sites; etomidate 1. Introduction GABAA receptors are pentameric anion channels heavily expressed in the mammalian central nervous system. They are formed of five homologous subunits, encoded by 19 different genes. Despite the significant subtype heterogeneity, it is commonly accepted that the majority of receptors is formed of two α and β subunits and a single γ subunit. The subunits form a pentameric GABA gated ion pore, with subunit interfaces formed by a principal (or +) and a complementary (or ) face of each subunit as − Int. J. Mol. Sci. 2020, 21, 334; doi:10.3390/ijms21010334 www.mdpi.com/journal/ijms Int.Int. J. J. Mol. Mol. Sci. Sci. 20202020, ,2121, ,334 334 2 2of of 20 20 of each subunit as depicted in Figure 1. Treatments of epilepsy, anxiety states and sleep disorders, as welldepicted as anesthetics, in Figure1 .target Treatments GABA ofA receptors epilepsy, anxiety[1,2]. Among states andthese sleep drugs, disorders, benzodiazepines as well as anesthetics,are widely prescribedtarget GABA andA receptorsare known [1 to,2 ].exert Among their these effects drugs, via a benzodiazepinesbinding site on the are extracellular widely prescribed domain and of the are receptor.known to Specifically, exert their e fftheects binding via a binding site of site benzodiazepines on the extracellular is situated domain at of the the extracellular receptor. Specifically, domain the binding site of benzodiazepines is situated at the extracellular domain (ECD)- interface α+/γ (ECD)- interface α+/γ− (site 1, Figure 1) and homologous with the GABA binding site at the ECD-− (site 1, Figure1) and homologous with the GABA binding site at the ECD- β+/α interface [3,4]. β+/α− interface [3,4]. − BenzodiazepinesBenzodiazepines interact interact additionally additionally with with another another low low affinity affinity binding binding site site within within the the transmembranetransmembrane domain domain (TMD), (TMD), at at the the TMD-interface β+/+/α−α (site(site 3) 3) [5]. [5]. This This binding binding site site is is chiefly chiefly − knownknown as as the the site site of of action action for for the the clinically clinically used used drug drug etomidate, etomidate, an andd also also one one of of the the interaction interaction sites sites forfor propofol propofol [6,7]. [6,7]. BesidesBesides the the clinically clinically relevant relevant drugs, drugs, a a wide wide range range of of compounds compounds have have been been identified identified as as modulatorsmodulators of of the the GABA GABAAA receptorsreceptors [1,8–10]. [1,8–10]. FigureFigure 1. (a(a) )Schematic Schematic rendering rendering of of a a GABAA receptor.receptor. The The extracellular extracellular domain domain (ECD) (ECD) and and transmembranetransmembrane domain domain (TMD) (TMD) contai containn various various binding binding sites sites [11]. [11]. The The binding binding sites sites studied studied in in this this workwork are atat interfacesinterfaces formed formed by by a a principal principal (+ (+)) subunit subunit face face and and a complementary a complementary ( ) subunit(−) subunit face. face. The − Theshape shape resembles resembles vaguely vaguely a space a fillingspace renderingfilling rendering of the protein’s of the ECDprotein’s and TMD,ECD whileand TMD, intracellular while intracellulardomain (ICD) domain shape is(ICD) a purely shape schematic is a purely rendering schematic based rendering on more based remote on homologues.more remote homologues. (b) Schematic (planesb) Schematic through planes the ECDthrough (top) the and ECD TMD (top) (bottom) and TMD of (bottom) a canonical of a canonicalαβγ receptor, αβγ receptor, the GABA the sites GABA are indicated as dark grey ellipses. Site 1 (blue), the high affinity benzodiazepine site, is at the ECD– α+/γ sites are indicated as dark grey ellipses. Site 1 (blue), the high affinity benzodiazepine site, is at the− ECD–interface; α+/γ− site interface; 2 (blue), site which 2 (blue), confers which modulatory confers emoduffectslatory of pyrazoloquinolinone effects of pyrazoloquinolinone ligands is at the ligands ECD– α+/β interface, and site 3 (orange), the etomidate site [7] occurs twice and is located at the TMD– is at the− ECD– α+/β− interface, and site 3 (orange), the etomidate site [7] occurs twice and is located at β+/α interfaces below the GABA binding sites. the TMD–− β+/α− interfaces below the GABA binding sites. InIn a a previous previous work, work, we we identified identified a a series series of of indole indole derivatives, derivatives, sharing sharing pharmacophore pharmacophore features features with pyrazoloquinolinones (PQs), as GABA modulators (Figure2a) [ 12]. PQs are known to bind with with pyrazoloquinolinones (PQs), as GABAA A modulators (Figure 2a) [12]. PQs are known to bind withhigh high affinity affinity at the at BZthe siteBZ site (site (site 1) and1) and to to modulate modulate the the receptors receptors via via the the interactioninteraction withwith a second second binding site of the ECD, at the interface between α+ and β (site 2) [13]. An interaction of PQs with binding site of the ECD, at the interface between α+ and β−− (site 2) [13]. An interaction of PQs with sitesite 3 3 has has also also been been reported reported [14] [14] For For this this study, study, we selected the most efficacious efficacious compound of of the the previouslypreviously published library, MTI163 (Figure2 2).). Int. J. Mol. Sci. 2020, 21, 334 3 of 20 Int. J. Mol. Sci. 2020, 21, 334 3 of 20 FigureFigure 2. (a 2.) Structure(a) Structure of MTI163; of MTI163; (b) structure(b) structure of DCBS192; of DCBS192; (c) pharmacophores (c) pharmacophores of indole of indole derivatives derivatives (left)(left) and and of PQs of PQs (right); (right); (d) superposition(d) superposition of pharmacophores of pharmacophores of PQ of andPQ and indole indole derivatives. derivatives. Panels Panels c c andand d depict d depict the pharmacophoresthe pharmacophores already already discussed discussed in [12 in]. [12]. The high pharmacophoric overlap of the indole derivatives in comparison with the PQs (Figure2c,d) The high pharmacophoric overlap of the indole derivatives in comparison with the PQs (Figure suggests similar binding behavior. We therefore considered the three possible binding sites as object 2c,d) suggests similar binding behavior. We therefore considered the three possible binding sites as of our studies: site 1 (ECD- α+/γ ) site 2 (ECD- α+/β ), and site 3 (TMD- β+/α ). To investigate object of our studies: site 1 (ECD-− α+/γ−) site 2 (ECD-− α+/β−), and site 3 (TMD- −β+/α−). To investigate the putative binding and mechanism of action of the new scaffold at these three sites, we performed the putative binding and mechanism of action of the new scaffold at these three sites, we performed functional and mutational studies, as well as radioligand displacement assays. Computational docking functional and mutational studies, as well as radioligand displacement assays. Computational studies complement the experimental findings in the context of the recently published cryo EM docking studies complement the experimental findings in the context of the recently published cryo structure with the PDB identifier 6HUP [4], which features diazepam in binding sites 1 and 3. EM structure with the PDB identifier 6HUP [4], which features diazepam in binding sites 1 and 3. 2. Results 2. Results 2.1. Chemistry 2.1. Chemistry The indole derivative MTI163 was synthesized as previously described [12].