A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C.

The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two ß-, and one γ2-subunit, whereas the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α5-subunit-containing GABAA receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.

Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA Receptors.

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular αx+/γ2- ( x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/ß- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure-activity relationship models for benzodiazepine site activity. Considering all possible α+/ß- and α+/γ- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2- site with the homologous α1+/ß3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2- subunit into the ß3- subunit, resulting in a quadruple mutant ß3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1ß3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/ß- sites.

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.

Targeting the γ-Aminobutyric Acid A Receptor α4 Subunit in Airway Smooth Muscle to Alleviate Bronchoconstriction.

We previously demonstrated that airway smooth muscle (ASM) cells express γ-aminobutyric acid A receptors (GABA(A)Rs), and that GABA(A)R agonists acutely relax ASM. Among the GABA(A)R α subunits, human ASM cells express only α4 and α5, providing the opportunity for selective pharmacologic targeting. Novel GABA(A)R-positive allosteric modulators designed for enhanced α4/α6 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 (EC3) of GABA [EC3]-induced currents in oocytes expressing α4 or α6 subunits (along with ß3 and γ2) compared with other α 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 α4 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 albuterol-mediated 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 α4 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 α1-3 subunit-containing GABA(A)Rs in the brain.

Metal-assisted synthesis of unsymmetrical magnolol and honokiol analogs and their biological assessment as GABAA receptor ligands.

We present the synthesis of new derivatives of natural products magnolol (1) and honokiol (2) and their evaluation as allosteric ligands for modulation of GABAA receptor activity. New derivatives were prepared via metal assisted cross-coupling reactions in two consecutive steps. Compounds were tested by means of two-electrode voltage clamp electrophysiology at the α1ß2γ2 receptor subtype at low GABA concentrations. We have identified several compounds enhancing GABA induced current (IGABA) in the range similar or even higher than the lead structures. At 3µM, compound 8g enhanced IGABA by factor of 443, compared to 162 and 338 of honokiol and magnolol, respectively. Furthermore, 8g at EC10-20 features a much bigger window of separation between the α1ß2γ2 and the α1ß1γ2 subtypes compared to honokiol, and thus improved subtype selectivity.

A pentasymmetric open channel blocker for Cys-loop receptor channels.

γ-Aminobutyric acid type A receptors (GABAA receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl- ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP-) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α1V256-α1T261 in the rat recombinant GABAA receptor to be important for PCCP- action. Introduction of positive charges into M2 increased the affinity for PCCP- while PCCP- prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP-, among them the Drosophila RDL GABAA receptor carrying an insecticide resistance mutation, suggesting that PCCP- could serve as an insecticide.

Accelerated discovery of novel benzodiazepine ligands by experiment-guided virtual screening.

High throughput discovery of ligand scaffolds for target proteins can accelerate development of leads and drug candidates enormously. Here we describe an innovative workflow for the discovery of high affinity ligands for the benzodiazepine-binding site on the so far not crystallized mammalian GABAA receptors. The procedure includes chemical biology techniques that may be generally applied to other proteins. Prerequisites are a ligand that can be chemically modified with cysteine-reactive groups, knowledge of amino acid residues contributing to the drug-binding pocket, and crystal structures either of proteins homologous to the target protein or, better, of the target itself. Part of the protocol is virtual screening that without additional rounds of optimization in many cases results only in low affinity ligands, even when a target protein has been crystallized. Here we show how the integration of functional data into structure-based screening dramatically improves the performance of the virtual screening. Thus, lead compounds with 14 different scaffolds were identified on the basis of an updated structural model of the diazepam-bound state of the GABAA receptor. Some of these compounds show considerable preference for the α3ß2γ2 GABAA receptor subtype.