GABA A receptorsmediatetheactionofmanyclinicallyimportantdrugsinteractingwithdifferentbindingsites.Forsomepotentialbindingsites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular ␣1ϩ␤3Ϫ interface, which is homologous to the benzodiazepine binding site at the ␣1ϩ␥2Ϫ interface. On screening of Ͼ100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3Ϫc]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at ␣1␤3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at ␣1␤3␥2 and ␣1␤3 receptors. By mutating pocket-forming amino acid residues at the ␣1ϩ and the ␤3Ϫ side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethiosulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect of CGS 9895. The inhibition was not caused by a general inactivation of GABA A receptors, because the GABA-enhancing effect of ROD 188 or the steroid ␣-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the ␣ and ␤ subunit types forming the interface. CGS 9895thusrepresentsthefirstprototypeofdrugsmediatingbenzodiazepine-likemodulatoryeffectsviathe␣ϩ␤ϪinterfaceofGABA A receptors. Sincesuchbindingsitesarepresentat␣␤,␣␤␥,and␣␤␦receptors,suchdrugswillhaveamuchbroaderactionthanbenzodiazepinesandmight become clinical important for the treatment of epilepsy.

Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABA A 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 un biased 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 benzodiazepinebinding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design. GABA A receptors (GABA A Rs) are the major inhibitory transmitter receptors in the brain and the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anesthetics and convulsants 1 . These receptors are chloride ion channels composed of five subunits that can belong to different subunit classes. A total of 19 GABA A receptor subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-© 2012 Nature America, Inc. All rights reserved. * margot.ernst@meduniwien.ac.at. author contributions 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. GABA A R 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. Competing financial interestsThe authors declare no competing financial interests. Additional informationSupplementary information and chemical compound information are available online at http://www.nature.com/ naturechemicalbiology/. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to M.E. Accession codes. PDB: the previously determined crystal structures for A. californica AChBP, L. stagnalis AChBP, mouse nAChR, a nematode glutamategated chloride channel, an nAChR structure and four AChBP structures are deposited under accession codes 2BYQ, 1UW6, 2QC1, 3RIF, 2BG9, 1I9B, 2BYN, 2BYR and 2BYS, respectively. Europe PMC Funders GroupAuthor Manuscript Nat Chem Biol. Author manuscript; available in PMC 2012 June 06. Europe PMC Funders Author ManuscriptsEurope PMC Fun...

BACKGROUND AND PURPOSEGABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo [4,3-c]quinolin-3(5H)-one) acts as a null modulator (antagonist) at the high affinity benzodiazepine binding site, but in addition elicits a strong enhancement of GABA-induced currents via a novel drug binding site at the extracellular a+b-interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the a1+b3-interface of GABAA receptors. EXPERIMENTAL APPROACHGABAA receptors were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. KEY RESULTSWe not only identified compounds with higher efficacy/potency than CGS 9895 for stimulating GABA-induced currents via the a1+b3-binding site, but also discovered compounds acting as null modulators at this site. Most of the compounds also acted as null modulators via the benzodiazepine binding site of GABAA receptors. But some of the positive allosteric modulators or null modulators exclusively exerted their action via the a+b-binding site. CONCLUSION AND IMPLICATIONSPyrazoloquinolinones and pyrazolopyridinones represent the first prototype of drug candidates mediating benzodiazepine like modulatory effects via the a+b-interface of GABAA receptors. The discovery of null modulators acting as inhibitors of the plus modulators provides a highly useful tool for the discovery of additional classes of compounds that can modulate GABAA receptors via this site, which may lead to novel therapeutic principles. LINKED ARTICLEThis article is accompanied by Varagic et al.,

BACKGROUND AND PURPOSEGABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo [4,3-c]quinolin-3(5H)-one) elicits a strong and subtype-dependent enhancement of GABA-induced currents via a novel drug-binding site at extracellular ax+by-(x = 1-6, y = 1-3) interfaces. Here, we investigated 16 structural analogues of CGS 9895 for their ability to modulate GABA-induced currents of various GABAA receptor subtypes. EXPERIMENTAL APPROACHRecombinant GABAA receptor subtypes were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. KEY RESULTSMost of the compounds investigated were able to modulate GABA-induced currents of ab and abg receptors to a comparable extent, suggesting that the effect of these drugs is not dependent on the benzodiazepine site of GABAA receptors. Steric hindrance experiments demonstrated that these compounds exert their action predominantly via the ax+by-(x = 1-6, y = 1-3) interfaces. Whereas some compounds are unselectively modulating a broad range of receptor subtypes, other compounds feature remarkable functional selectivity for the a6b3g2 receptor, or behave as null modulators at some receptor subtypes investigated. CONCLUSION AND IMPLICATIONSPyrazoloquinolinones and pyrazolopyridinones represent the first prototypes of drugs exerting benzodiazepine-like modulatory effects via the a+b-interface of GABAA receptors. The discovery of modulators with functional subtype selectivity at this class of binding sites provides a highly useful tool for the investigation of a6b2/3g2 receptor function, and may lead to novel therapeutic principles. LINKED ARTICLE

GABAA receptors are ligand‐gated chloride channels composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to a multiplicity of GABAA receptor subtypes with distinct subunit composition; regional, cellular and subcellular distribution; and pharmacology. Most of these receptors are composed of two α, two β and one γ2 subunits. GABAA receptors are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anaesthetics and convulsants. Whereas GABA acts at the two extracellular β+α‐ interfaces of GABAA receptors, the allosteric modulatory benzodiazepines interact with the extracellular α+γ2‐ interface. In contrast, barbiturates, neuroactive steroids and anaesthetics seem to interact with solvent accessible pockets in the transmembrane domain. Several benzodiazepine site ligands have been identified that selectively interact with GABAA receptor subtypes containing α2βγ2, α3βγ2 or α5βγ2 subunits. This indicates that the different α subunit types present in these receptors convey sufficient structural differences to the benzodiazepine binding site to allow specific interaction with certain benzodiazepine site ligands. Recently, a novel drug binding site was identified at the α+β‐ interface. This binding site is homologous to the benzodiazepine binding site at the α+γ2‐ interface and is thus also strongly influenced by the type of α subunit present in the receptor. Drugs interacting with this binding site cannot directly activate but only allosterically modulate GABAA receptors. The possible importance of such drugs addressing a spectrum of receptor subtypes completely different from that of benzodiazepines is discussed.

γ-Aminobutyric acid type A (GABAA) receptors are pentameric GABA-gated chloride channels that are, in mammalians, drawn from a repertoire of 19 different genes, namely α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3. The existence of this wide variety of subunits as well as their diverse assembly into different subunit compositions result in miscellaneous receptor subtypes. In combination with the large number of known and putative allosteric binding sites, this leads to a highly complex pharmacology. Recently, a novel binding site at extracellular α+/β− interfaces was described as the site of modulatory action of several pyrazoloquinolinones. In this study we report a highly potent ligand from this class of compounds with pronounced β1-selectivity that mainly lacks α-subunit selectivity. It constitutes the most potent β1-selective positive allosteric modulatory ligand with known binding site. In addition, a proof of concept pyrazoloquinolinone ligand lacking the additional high affinity interaction with the benzodiazepine binding site is presented. Ultimately, such ligands can be used as invaluable molecular tools for the detection of β1-containing receptor subtypes and the investigation of their abundance and distribution.

Background and PurposeThe GABAA receptors are ligand‐gated ion channels, which play an important role in neurotransmission. Their variety of binding sites serves as an appealing target for many clinically relevant drugs. Here, we explored the functional selectivity of modulatory effects at specific extracellular α+/β− interfaces, using a systematically varied series of pyrazoloquinolinones.Experimental ApproachRecombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABA‐elicited currents by the newly synthesized and reference compounds were investigated by the two‐electrode voltage clamp method.Key ResultsWe identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at α6β3γ2 GABAA receptors with nearly no residual activity at the other αxβ3γ2 (x = 1–5) subtypes. This modulation was independent of affinity for α+/γ− interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular α+/β− interfaces.Conclusion and ImplicationsThese results constitute a major step towards a potential selective positive modulation of certain α6‐containing GABAA receptors, which might be useful to elicit their physiological role. Furthermore, these studies pave the way towards insights into molecular principles that drive positive versus negative allosteric modulation of specific GABAA receptor isoforms.

We previously demonstrated that airway smooth muscle (ASM) cells express g-aminobutyric acid A receptors (GABA A Rs), and that GABA A R agonists acutely relax ASM. Among the GABA A R a subunits, human ASM cells express only a4 and a5, providing the opportunity for selective pharmacologic targeting. Novel GABA A R-positive allosteric modulators designed for enhanced a4/a6 subunit selectivity were synthesized using iterative computational analyses (CMD-45 and XHe-III-74). Studies using oocyte heterologous expression systems confirmed that CMD-45 and XHe-III-74 led to significantly greater augmentation of currents induced by a 3% maximal effective concentration (EC 3 ) of GABA [EC 3 ]-induced currents in oocytes expressing a4 or a6 subunits (along with b3 and g2) compared with other a subunits. CMD-45 and XHe-III-74 also led to greater ex vivo relaxation of contracted wild-type mouse tracheal rings compared with tracheal rings from GABA A R a4 subunit (Gabra4) knockout mice. Furthermore, CMD-45 and XHe-III-74 significantly relaxed precontracted human ASM ex vivo, and, at a low concentration, both ligands led to a significant leftward shift in albuterolmediated ASM relaxation. In vivo, inhaled XHe-III-74 reduced respiratory system resistance in an asthmatic mouse model. Pretreatment of human ASM cells with CMD-45 and XHe-III-74 inhibited histamine-induced increases in intracellular calcium concentrations in vitro, an effect that was lost when calcium was omitted from the extracellular buffer, suggesting that inhibition of calcium influx due to alterations in plasma membrane potential may play a role in the mechanism of ASM relaxation. Selective targeting of the GABA A R a4 subunit with inhaled ligands may be a novel therapeutic pathway to treat bronchoconstriction, while avoiding sedative central nervous system effects, which are largely mediated by a1-3 subunit-containing GABA A Rs in the brain.