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.

Towards functional selectivity for α6ß3γ2 GABAA receptors: a series of novel pyrazoloquinolinones.

BACKGROUND AND PURPOSE: The 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+/ß- interfaces, using a systematically varied series of pyrazoloquinolinones. EXPERIMENTAL APPROACH: Recombinant 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 RESULTS: We identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at α6ß3γ2 GABAA receptors with nearly no residual activity at the other αxß3γ2 (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+/ß- interfaces. CONCLUSION AND IMPLICATIONS: These 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.

Molecular tools for GABAA receptors: High affinity ligands for ß1-containing subtypes.

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, ß1-3, 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+/ß- 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 ß1-selectivity that mainly lacks α-subunit selectivity. It constitutes the most potent ß1-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 ß1-containing receptor subtypes and the investigation of their abundance and distribution.

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.

Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: the approximated activation of receptor subtypes may explain behavioral effects.

Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10mg/kg dose of the novel ligand and 2mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands.

Unexpected Properties of δ-Containing GABAA Receptors in Response to Ligands Interacting with the α+ ß- Site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are the targets of many clinically important drugs, which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. Recently, we described an allosteric modulation occurring upon binding of pyrazoloquinolinones to a novel binding site at the extracellular α+ ß- interface. Here, we investigated the effect of 4-(8-methoxy-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (the pyrazoloquinolinone LAU 177) at several αß, αßγ and αßδ receptor subtypes. LAU 177 enhanced GABA-induced currents at all receptors investigated, and the extent of modulation depended on the type of α and ß subunits present within the receptors. Whereas the presence of a γ2 subunit within αßγ2 receptors did not dramatically change LAU 177 induced modulation of GABA currents compared to αß receptors, we observed an unexpected threefold increase in modulatory efficacy of this compound at α1ß2,3δ receptors. Steric hindrance experiments as well as inhibition by the functional α+ ß- site antagonist LAU 157 indicated that the effects of LAU 177 at all receptors investigated were mediated via the α+ ß- interface. The stronger enhancement of GABA-induced currents by LAU 177 at α1ß3δ receptors was not observed at α4,6ß3δ receptors. Other experiments indicated that this enhancement of modulatory efficacy at α1ß3δ receptors was not observed with another α+ ß- modulator, and that the efficacy of modulation by α+ ß- ligands is influenced by all subunits present in the receptor complex and by structural details of the respective ligand.

Insights into functional pharmacology of α1 GABA(A) receptors: how much does partial activation at the benzodiazepine site matter?

RATIONALE: Synthesis of ligands inactive or with low activity at α1 GABA(A) receptors has become the key concept for development of novel, more tolerable benzodiazepine (BZ)-like drugs. WYS8, a remarkably (105 times) α1-subtype selective partial positive modulator, may serve as a pharmacological tool for refining the role of α1 GABA(A) receptors in mediation of BZs' effects. OBJECTIVES: Here, the effects of WYS8 on GABA-induced currents and on diazepam-induced potentiation of recombinant BZ-sensitive GABA(A) receptors were studied in more detail. In addition, the behavioral profile of WYS8 (0.2, 1, and 10 mg/kg i.p.), on its own and in combination with diazepam, was tested in the spontaneous locomotor activity, elevated plus maze, grip strength, rotarod, and pentylenetetrazole tests. RESULTS: WYS8, applied at an in vivo attainable concentration of 100 nM, reduced the stimulation of GABA currents by 1 µM diazepam by 57 % at α1ß3γ2, but not at α2ß3γ2, α3ß3γ2, or α5ß3γ2 GABA(A) receptors. The administration of WYS8 alone induced negligible behavioral consequences. When combined with diazepam, WYS8 caused a reduction in sedation, muscle relaxation, and anticonvulsant activity, as compared with this BZ alone, whereas ataxia was preserved, and the anxiolytic effect of 2 mg/kg diazepam was unmasked. CONCLUSIONS: Hence, a partial instead of full activation at α1 GABA(A) receptors did not necessarily result in the attenuation of the effects assumed to be mediated by activation of these receptors, or in the full preservation of the effects mediated by activation of other GABA(A) receptors. Thus, the role of α1 GABA(A) receptors appears more complex than that proposed by genetic studies.

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

BACKGROUND AND PURPOSE: 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+ß- interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the α1+ß3- interface of GABAA receptors. EXPERIMENTAL APPROACH: GABAA receptors were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. KEY RESULTS: We not only identified compounds with higher efficacy/potency than CGS 9895 for stimulating GABA-induced currents via the α1+ß3-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 α+ß- binding site. CONCLUSION AND IMPLICATIONS: Pyrazoloquinolinones and pyrazolopyridinones represent the first prototype of drug candidates mediating benzodiazepine like modulatory effects via the α+ß-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.

Subtype selectivity of α+ß- site ligands of GABAA receptors: identification of the first highly specific positive modulators at α6ß2/3γ2 receptors.

BACKGROUND AND PURPOSE: 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+ßy- (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 APPROACH: Recombinant GABAA receptor subtypes were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. KEY RESULTS: Most of the compounds investigated were able to modulate GABA-induced currents of αß and αßγ 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 αx+ßy- (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 α6ß3γ2 receptor, or behave as null modulators at some receptor subtypes investigated. CONCLUSION AND IMPLICATIONS: Pyrazoloquinolinones and pyrazolopyridinones represent the first prototypes of drugs exerting benzodiazepine-like modulatory effects via the α+ß- 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 α6ß2/3γ2 receptor function, and may lead to novel therapeutic principles.

Benzodiazepine-induced spatial learning deficits in rats are regulated by the degree of modulation of α1 GABA(A) receptors.

Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABAA receptors, containing the α1, α2, α3 or α5 subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α1-subunit affinity-selective antagonist ß-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α1-subunit selective ligand-WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or ß-CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α1-subtype selective weak partial positive modulator (40% potentiation at 100nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with ß-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α1 subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.

Search for α3ß2/3γ2 subtype selective ligands that are stable on human liver microsomes.

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA(A)) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2ß(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100mg/kg and was devoid of sedative properties.

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.

A novel GABA(A) receptor pharmacology: drugs interacting with the α(+) ß(-) interface.

GABA(A) 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 GABA(A) receptor subtypes with distinct subunit composition; regional, cellular and subcellular distribution; and pharmacology. Most of these receptors are composed of two α, two ß and one γ2 subunits. GABA(A) 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 ß(+) α(-) interfaces of GABA(A) 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 GABA(A) receptor subtypes containing α2ßγ2, α3ßγ2 or α5ßγ2 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(+) ß(-) 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 GABA(A) receptors. The possible importance of such drugs addressing a spectrum of receptor subtypes completely different from that of benzodiazepines is discussed.

The GABAA receptor alpha+beta- interface: a novel target for subtype selective drugs.

GABA(A) 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+ß3- 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 α1ß3 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 α1ß3γ2 and α1ß3 receptors. By mutating pocket-forming amino acid residues at the α1+ and the ß3- 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 α-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the α and ß subunit types forming the interface. CGS 9895 thus represents the first prototype of drugs mediating benzodiazepine-like modulatory effects via the α+ß- interface of GABA(A) receptors. Since such binding sites are present at αß, αßγ, and αßδ receptors, such drugs will have a much broader action than benzodiazepines and might become clinical important for the treatment of epilepsy.