Margot Ernst

Comparative modeling of GABAA receptors: limits, insights, future developments

GABA(A) receptors are chloride ion channels that mediate fast synaptic transmission and belong to a superfamily of pentameric ligand-gated ion channels. The recently published crystal structure of the acetylcholine binding protein can be used as a template for comparative modeling of the extracellular domain of GABA(A) receptors. In this commentary, difficulties with comparative modeling at low sequence identity are discussed, the degree of structural conservation to be expected within the superfamily is analyzed and numerical estimates of model uncertainties in functional regions are provided. Topography of the binding sites at subunit-interfaces is examined and possible targets for rational mutagenesis studies are suggested. Allosteric motions are considered and a mechanism for mediation of positive cooperativity at the benzodiazepine site is proposed.

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 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 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.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

GABAA receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABAA receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABAA receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

The GABAA Receptor α+β− Interface: A Novel Target for Subtype Selective Drugs

GABAA receptors mediate the action of many clinically important drugs interacting with different binding sites. For some potential binding sites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular α1+β3− interface, which is homologous to the benzodiazepine binding site at the α1+γ2− interface. On screening of >100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3−c]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at α1β3 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 α1β3γ2 and α1β3 receptors. By mutating pocket-forming amino acid residues at the α1+ and the β3− 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 GABAA receptors, because the GABA-enhancing effect of ROD 188 or the steroid α-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the α and β subunit types forming the interface. CGS 9895 thus represents the first prototype of drugs mediating benzodiazepine-like modulatory effects via the α+β− interface of GABAA receptors. Since such binding sites are present at αβ, αβγ, and αβδ receptors, such drugs will have a much broader action than benzodiazepines and might become clinical important for the treatment of epilepsy.

A novel GABAA receptor pharmacology: drugs interacting with the α+β− interface

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.

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators.

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the γ-aminobutyric acid type A (GABAA) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABAA receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABAA receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly α1H101C and also three other mutants (α1G157C, α1V202C, and α1V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (–NCS) replaces the azide group (–N3) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, α1H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.

Subtype selectivity of α+β− site ligands of GABAA receptors: identification of the first highly specific positive modulators at α6β2/3γ2 receptors

GABAA 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 αx+βy− (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.

Structural Studies of GABAA Receptor Binding Sites: Which Experimental Structure Tells us What?

Atomic resolution structures of cys-loop receptors, including one of a γ-aminobutyric acid type A receptor (GABAA receptor) subtype, allow amazing insights into the structural features and conformational changes that these pentameric ligand-gated ion channels (pLGICs) display. Here we present a comprehensive analysis of more than 30 cys-loop receptor structures of homologous proteins that revealed several allosteric binding sites not previously described in GABAA receptors. These novel binding sites were examined in GABAA receptor homology models and assessed as putative candidate sites for allosteric ligands. Four so far undescribed putative ligand binding sites were proposed for follow up studies based on their presence in the GABAA receptor homology models. A comprehensive analysis of conserved structural features in GABAA and glycine receptors (GlyRs), the glutamate gated ion channel, the bacterial homologs Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus GLIC, and the serotonin type 3 (5-HT3) receptor was performed. The conserved features were integrated into a master alignment that led to improved homology models. The large fragment of the intracellular domain that is present in the structure of the 5-HT3 receptor was utilized to generate GABAA receptor models with a corresponding intracellular domain fragment. Results of mutational and photoaffinity ligand studies in GABAA receptors were analyzed in the light of the model structures. This led to an assignment of candidate ligands to two proposed novel pockets, candidate binding sites for furosemide and neurosteroids in the trans-membrane domain were identified. The homology models can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo-EM microscopic structures of the closest homologs that were solved in different conformational states provided important insights into structural rearrangements of binding sites during conformational transitions. The impact of structural variation and conformational motion on the shape of the investigated binding sites was analyzed. Rules for best template and alignment choice were obtained and can generally be applied to modeling of cys-loop receptors. Overall, we provide an updated structure based view of ligand binding sites present in GABAA receptors.

Molecular analysis of the site for 2-arachidonylglycerol (2-AG) on the β2 subunit of GABAA receptors

2‐arachidonyl glycerol (2‐AG) allosterically potentiates GABAA receptors via a binding site located in transmembrane segment M4 of the β2 subunit. Two amino acid residues have been described that are essential for this effect. With the aim to further describe this potential drug target, we performed a cysteine scanning of the entire M4 and part of M3. All four residues in M4 affecting the potentiation here and the two already identified residues locate to the same side of the α‐helix. This side is exposed to M3, where further residues were identified. From the fact that the important residues span > 18 Å, we conclude that the hydrophobic tail of the bound 2‐AG molecule must be near linear and that the site mainly locates to the inner leaflet but stretches far into the membrane. The influence of the structure of the head group of the ligand molecule on the activity of the molecule was also investigated. We present a model of 2‐AG docked to the GABAA receptor.

Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β- interface.

GABAA 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 α+β− interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the α1+β3− interface of GABAA receptors.