Integrated workflow for the identification of new GABAA R positive allosteric modulators based on the in†silico screening with further in†vitro validation. Case study using Enamine's stock chemical space.

Numerous studies reported an association between GABAA R subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABAA R-diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1ß2γ2 with some Z-drugs (non-benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre-filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1ß2γ2 GABAA receptors. Whole-cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABAA receptors. A High-throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon.

Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1ß2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/ß subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Anesthetic Binding Induced Motion of GABAA Receptors Revealed by Coarse-Grained Molecular Dynamics Simulations.

General anesthetics are indispensable in modern medicine because they induce a reversible loss of consciousness and sensation in humans. On the other hand, their molecular mechanisms of action have not yet been elucidated. Several studies have identified the main targets of some general anesthetics. The structures of γ-aminobutyric acid A (GABAA) receptors with the intravenous anesthetics such as propofol and etomidate have recently been determined. Although these anesthetic binding structures provide essential insights into the mechanism of action of anesthetics, the detailed molecular mechanism of how the anesthetic binding affects the Cl- permeability of GABAA receptors remains to be elucidated. In this study, we performed coarse-grained molecular dynamics simulations for GABAA receptors and analyzed the resulting simulation trajectories to investigate the effects of anesthetic binding on the motion of GABAA receptors. The results showed large structural fluctuations in GABAA receptors, correlations of motion between the amino acid residues, large amplitude motion, and autocorrelated slow motion, which were obtained by advanced statistical analyses. In addition, a comparison of the resulting trajectories in the presence or absence of the anesthetic molecules revealed a characteristic pore motion related to the gate-opening motion of GABAA receptors.

GABAA receptor-mediated seizure liabilities: a mixed-methods screening approach.

GABAA receptors, members of the pentameric ligand-gated ion channel superfamily, are widely expressed in the central nervous system and mediate a broad range of pharmaco-toxicological effects including bidirectional changes to seizure threshold. Thus, detection of GABAA receptor-mediated seizure liabilities is a big, partly unmet need in early preclinical drug development. This is in part due to the plethora of allosteric binding sites that are present on different subtypes of GABAA receptors and the critical lack of screening methods that detect interactions with any of these sites. To improve in silico screening methods, we assembled an inventory of allosteric binding sites based on structural data. Pharmacophore models representing several of the binding sites were constructed. These models from the NeuroDeRisk IL Profiler were used for in silico screening of a compiled collection of drugs with known GABAA receptor interactions to generate testable hypotheses. Amoxapine was one of the hits identified and subjected to an array of in vitro assays to examine molecular and cellular effects on neuronal excitability and in vivo locomotor pattern changes in zebrafish larvae. An additional level of analysis for our compound collection is provided by pharmacovigilance alerts using FAERS data. Inspired by the Adverse Outcome Pathway framework, we postulate several candidate pathways leading from specific binding sites to acute seizure induction. The whole workflow can be utilized for any compound collection and should inform about GABAA receptor-mediated seizure risks more comprehensively compared to standard displacement screens, as it rests chiefly on functional data.

Genetic interaction between GABRA1 and ERBB4 variants in the pathogenesis of genetic generalized epilepsy.

Epilepsy is a complex neurological disease that can be caused by both genetic and environmental factors. Many studies have been conducted to investigate the genetic risk variants and molecular mechanisms of epilepsy. Disruption of excitation-inhibition balance (E/I balance) is one of the widely accepted disease mechanisms of epilepsy. The maintenance of E/I balance is an intricate process that is governed by multiple proteins. Using whole exome sequencing (WES), we identified a novel GABRA1 c.448G>A (p.E150K) variant and ERBB4 c.1972A>T (p.I658F, rs190654033) variant in a Malaysian Chinese family with genetic generalized epilepsy (GGE). The GGE may be triggered by dysregulation of E/I balance mechanism. Segregation of the variants in the family was verified by Sanger sequencing. All family members with GGE inherited both variants. However, family members who carried only one of the variants did not show any symptoms of GGE. Both the GABRA1 and ERBB4 variants were predicted damaging by MutationTaster and CADD, and protein structure analysis showed that the variants had resulted in the formation of additional hydrogen bonds in the mutant proteins. GABRA1 variant could reduce the efficiency of GABAA receptors, and constitutively active ERBB4 receptors caused by the ERBB4 variant promote internalization of GABAA receptors. The interaction between the two variants may cause a greater disruption in E/I balance, which is more likely to induce a seizure. Nevertheless, this disease model was derived from a single small family, further studies are still needed to confirm the verifiability of the purported disease model.

Novel spiro[indene-1,2'-quinazolin]-4'(3'H)-one derivatives as potent anticonvulsant agents: One-pot synthesis, in vivo biological evaluation, and molecular docking studies.

A new series of spiro[indene-1,2'-quinazolin]-4'(3'H)-one derivatives 4a-m were synthesized via a one-pot method and evaluated for anticonvulsant activities using pentylenetetrazole (PTZ) and maximal electroshock (MES)-induced seizures. Obtained results demonstrated that these compounds have not anticonvulsant activity in PTZ test while are active in the MES test. Among the synthesized compounds, the best anticonvulsant activity was obtained with compound 4h. This compound also was not neurotoxic. Given that the title new compounds have the pharmacophore requirement for benzodiazepine (BZD) receptor agonist, the most potent compound was assayed in vivo and in silico as BZD receptor agonist. After treatment with flumazenil as a standard BZD receptor antagonist, anticonvulsant activity of compound 4h decreased. Therefore, the involvement of BZD receptors in anticonvulsant activity of this compound confirmed. Furthermore, docking study of compound 4h in the BZD-binding site of GABAA receptor confirmed that this compound interacted with the important residues.

Novel alpha6 preferring GABA-A receptor ligands based on loreclezole.

The family of GABA-A receptors contains nineteen mammalian subunits from which pentameric, GABA gated anion channels are assembled. The subunit encoded by the GABRA6 gene is highly expressed in the cerebellum and the receptors to which it contributes have recently been demonstrated to be a promising candidate as a novel drug target. Here we examined a series of loreclezole derivatives for potentially selective action at α6ß3γ2 receptors with the help of computational methods and functional testing with the two-electrode voltage clamp technique. The synthetic routes to some previously published ligands were improved, and a new derivative was synthesized based on computational docking results. This new loreclezole derivative, [3-(2-chloro-4-methylphenyl)-3-methylbutanenitrile (40)], was shown to display stronger modulatory action in concatenated α6ß3γ2 receptors compared to their α1ß3γ2 counterpart. The hypothetical bound state structure provides valuable guidance for future design of selective therapeutics.

Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature.

Several pyrazoloquinolinone (PQ) ligands were recently discovered as functionally selective positive modulators at the PQ site of α6-containing GABAA receptors. PQs are also neutral modulators at the benzodiazepine site. We assessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of male Sprague-Dawley rats. An excellent behavioural safety profile of all tested PQs was demonstrated in the spontaneous locomotor activity, rotarod, loss of righting reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029 and its analogues prevented the ataxic effects of the benzodiazepine, as assessed in the rotarod test and during monitoring of rat locomotor activity after awakening from the loss of righting reflex. Published electrophysiological profiles of PQ ligands imply that positive modulation elicited at α6-GABAA receptors that contain the γ2 and δ subunit, rather than their neutral modulatory action at the benzodiazepine site, may prevent the ataxic action of diazepam. Thus, PZ-II-029 and its deuterated analogues are not prone to untoward interactions with benzodiazepines and may indeed completely abolish their ataxic action, seen at therapeutic, and especially toxic concentrations.

Positive allosteric modulators of GABAA receptor restore chloride current from blockade by competitive antagonists in a ligand-dependent manner.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter. GABA receptor type A (GABAAR) possesses binding sites for a large group of pharmacological agents which are supposed to interact allosterically with each other. The aim of this work was to study the interaction between the positive allosteric modulators (PAMs) and the competitive antagonists of GABAARs. The GABA-induced chloride current (IGABA) was measured in isolated Purkinje cells of rat cerebellum using the patch-clamp technique. PAMs, neurosteroid allopregnanolone (Allo) and zolpidem (Zolp), a drug that positively modulates the GABAAR through interaction with the benzodiazepine (BDZ) site, doubled the IGABA amplitude in the control solution. Competitive antagonist of GABAARs, bicuculline (Bic, 5 µM) blocked the IGABA by 90%. The addition of 1 µM Allo or 0.5 µM Zolp to the Bic solution caused an unblocking effect, so that the IGABA amplitude increased 10 and 4 times from control value, correspondingly. This unblocking effect developed slowly, as evidenced by a threefold increase in the current rise time. Competitive antagonist of GABAARs, gabazine (GBZ, 0.5 µM) blocked the IGABA by 87%. The addition of 1 µM Allo to the GBZ solution caused an unblocking effect, so that the IGABA amplitude increased 7-fold. However, the addition of 0.5 µM Zolp to the GBZ solution did not cause an unblocking effect. So, Allo appeared to have a stronger unblocking potential than Zolp, and Bic binding site showed a higher sensitivity to the action of unblocking PAMs than GBZ binding site. The results indicate for the first time the existence of an allosteric relationship between the sites binding PAMs and the competitive antagonists of GABAAR.

Activation of the Rat α1ß2ε GABAA Receptor by Orthosteric and Allosteric Agonists.

GABAA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABAA subunits cloned to date. GABAA receptors containing the ε subunit exhibit a significant degree of constitutive activity and have been suggested to be unresponsive to allosteric agents. In this study, we have characterized the functional properties of the rat α1ß2ε GABAA receptor. We confirm that the α1ß2ε receptor exhibits a higher level of constitutive activity than typical of GABAA receptors and show that it is inefficaciously activated by the transmitter and the allosteric agonists propofol, pentobarbital, and allopregnanolone. Manipulations intended to alter ε subunit expression and receptor stoichiometry were largely without effect on receptor properties including sensitivity to GABA and allosteric agonists. Surprisingly, amino acid substitutions at the conserved 9' and 6' positions in the second transmembrane (TM2) domain in the ε subunit did not elicit the expected functional effects of increased constitutive activity and resistance to the channel blocker picrotoxin, respectively. We tested the accessibility of TM2 residues mutated to cysteine using the cysteine-modifying reagent 4-(hydroxymercuri)benzoic acid and found a unique pattern of water-accessible residues in the ε subunit.

Unravelling the Turn-On Fluorescence Mechanism of a Fluorescein-Based Probe in GABAA Receptors.

GABAA (γ-aminobutyric acid type A) receptors are ligand-gated ion channels mediating fast inhibitory transmission in the mammalian brain. Here we report the molecular and electronic mechanism governing the turn-on emission of a fluorescein-based imaging probe able to target the human GABAA receptor. Multiscale calculations evidence a drastic conformational change of the probe from folded in solution to extended upon binding to the receptor. Intramolecular ππ-stacking interactions present in the folded probe are responsible for quenching fluorescence in solution. In contrast, unfolding within the GABAA receptor changes the nature of the bright excited state triggering emission. Remarkably, this turn-on effect only manifests for the dianionic prototropic form of the imaging probe, which is found to be the strongest binder to the GABAA receptor. This study is expected to assist the design of new photoactivatable screening tools for allosteric modulators of the GABAA receptor.

Differential assembly diversifies GABAA receptor structures and signalling.

Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits1,2 and can be modulated by essential medicines including general anaesthetics and benzodiazepines3. Human GABAAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABAARs assembled from α4, ß3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors4-7. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data8,9, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABAARs.

Distinct Functional Alterations and Therapeutic Options of Two Pathological De Novo Variants of the T292 Residue of GABRA1 Identified in Children with Epileptic Encephalopathy and Neurodevelopmental Disorders.

Mutations of GABAAR have reportedly led to epileptic encephalopathy and neurodevelopmental disorders. We have identified a novel de novo T292S missense variant of GABRA1 from a pediatric patient with grievous global developmental delay but without obvious epileptic activity. This mutation coincidentally occurs at the same residue as that of a previously reported GABRA1 variant T292I identified from a pediatric patient with severe epilepsy. The distinct phenotypes of these two patients prompted us to compare the impacts of the two mutants on the receptor function and to search for suitable therapeutics. In this study, we used biochemical techniques and patch-clamp recordings in HEK293 cells overexpressing either wild-type or mutated rat recombinant GABAARs. We found that the α1T292S variant significantly increased GABA-evoked whole-cell currents, shifting the dose-response curve to the left without altering the maximal response. In contrast, the α1T292I variant significantly reduced GABA-evoked currents, shifting the dose-response curve to the right with a severely diminished maximum response. Single-channel recordings further revealed that the α1T292S variant increased, while the α1T292I variant decreased the GABAAR single-channel open time and open probability. Importantly, we found that the T292S mutation-induced increase in GABAAR function could be fully normalized by the negative GABAAR modulator thiocolchicoside, whereas the T292I mutation-induced impairment of GABAAR function was largely rescued with a combination of the GABAAR positive modulators diazepam and verapamil. Our study demonstrated that α1T292 is a critical residue for controlling GABAAR channel gating, and mutations at this residue may produce opposite impacts on the function of the receptors. Thus, the present work highlights the importance of functionally characterizing each individual GABAAR mutation for ensuring precision medicine.

GABRB2, a key player in neuropsychiatric disorders and beyond.

The GABA receptors represent the main inhibitory system in the central nervous system that ensure synaptogenesis, neurogenesis, and the regulation of neuronal plasticity and learning. GABAA receptors are pentameric in structure and belong to the Cys-loop superfamily. The GABRB2 gene, located on chromosome 5q34, encodes the ß2 subunit that combines with the α and γ subunits to form the major subtype of GABAA receptors, which account for 43% of all GABAA receptors in the mammalian brain. Each subunit probably consists of an extracellular N-terminal domain, four membrane-spanning segments, a large intracellular loop between TM3 and TM4, and an extracellular C-terminal domain. Alternative splicing of the RNA transcript of the GABRB2 gene gives rise at least to four long and short isoforms with dissimilar electrophysiological properties. Furthermore, GABRB2 is imprinted and subjected to epigenetic regulation and positive selection. It has been associated with schizophrenia first in Han Chinese, and subsequently validated in other populations. Gabrb2 knockout mice also exhibited schizophrenia-like behavior and neuroinflammation that were ameliorated by the antipsychotic drug risperidone. GABRB2 was also associated with other neuropsychiatric disorders including bipolar disorder, epilepsy, autism spectrum disorder, Alzheimer's disease, frontotemporal dementia, substance dependence, depression, internet gaming disorder, and premenstrual dysphoric disorder. Recently, it has been postulated that GABRB2 might be a potential marker for different cancer types. As GABRB2 has a pivotal role in the central nervous system and is increasingly recognized to contribute to human diseases, further understanding of its structure and function may expedite the generation of new therapeutic approaches.

Design, synthesis, and preliminary pharmacological evaluation of novel thiazolidinone derivatives as potential benzodiazepine agonists.

Thiazolidinones are well-known heterocycles that demonstrate promising biological effects such as anticonvulsant activity. Hybridization of these chemicals with scaffold, which has necessary pharmacophores for binding to the benzodiazepine receptors, can prompt a novel structure possessing extensive anticonvulsant effects. In this study, novel derivatives of thiazolidinone as new benzodiazepine agonists were designed, synthesized, and biologically evaluated. Compound 5h, 4-chloro-2-(2-fluorophenoxy)-N-(4-oxo-2-(p-tolyl)thiazolidin-3-yl)benzamide, exhibited considerable anticonvulsant activity, proper sedative-hypnotic effect, no memory impairment, and no muscle relaxant effect. The pharmacological effects of the designed compounds were antagonized by flumazenil, which confirmed the benzodiazepine receptors' involvement in their biological effects. Based on in silico calculations of ADME properties of our novel compounds, they could be active oral agents potentially. In this study, we designed novel structures by the hybridization of thiazolidinone moiety with scaffold which has necessary pharmacophores for binding to the benzodiazepine receptors. The results are very promising for developing new lead compounds as benzodiazepine agonists possess anticonvulsant effects.

The Sulfated Steroids Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate Inhibit the α1ß3γ2L GABAA Receptor by Stabilizing a Novel Nonconducting State.

The GABAA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α1ß3γ2L GABAA receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABAA receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state.

Neurosteroid Modulation of GABAA Receptor Function by Independent Action at Multiple Specific Binding Sites.

Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epiallopregnanolone (3ß-OH) are both prevalent in the mammalian brain and produce opposite effects on GABAA receptor function, acting as positive and negative allosteric modulators, respectively. This Perspective provides a model to explain the actions of 3α-OH and 3ß-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABAA receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.

Synthesis and Pharmacological Evaluation of Novel 2,3,4,5-tetrahydro[1,3]diazepino[1,2-a]benzimidazole Derivatives as Promising Anxiolytic and Analgesic Agents.

A number of novel 2,3,4,5-tetrahydro[1,3]diazepino[1,2-a]benzimidazole derivatives 2 were obtained by alkylation mainly in the 1H-tautomeric form of 2,3,4,5-tetrahydro[1,3]diazepino[1,2-a]benzimidazole or its 8,9-dimethyl-substituted analog 4-chlorobenzyl bromide, 4-chloroacetic acid fluoroanilide, and 4-tert-butylphenacyl bromide in neutral medium. Compounds 3 were cyclized and synthesized earlier with 11-phenacyl-substituted diazepino[1,2-a]benzimidazoles upon heating in conc. HBr. The chemical structures of the compounds were clarified by using the 1H Nuclear Magnetic Resonance Spectroscopy (1H-NMR) technique. Anxiolytic properties were evaluated using the elevated plus maze (EPM) and open field (OF) tests. The analgesic effect of compounds was estimated with the tail flick (TF) and hot plate (HP) methods. Besides, possible the influence of the test compounds on motor activities of the animals was examined by the Grid, Wire, and Rotarod tests. Compounds 2d and 3b were the most active due to their prominent analgesic and anxiolytic potentials, respectively. The results of the performed in silico analysis showed that the high anxiolytic activity of compound 3b is explained by the combination of a pronounced interaction mainly with the benzodiazepine site of the GABAA receptor with a prominent interaction with both the specific and allosteric sites of the 5-HT2A receptor.

In Silico Screening of Novel α1-GABAA Receptor PAMs towards Schizophrenia Based on Combined Modeling Studies of Imidazo [1,2-a]-Pyridines.

The ionotropic GABAA receptor (GABAAR) has been proven to be an important target of atypical antipsychotics. A novel series of imidazo [1,2-a]-pyridine derivatives, as selective positive allosteric modulators (PAMs) of α1-containing GABAARs with potent antipsychotic activities, have been reported recently. To better clarify the pharmacological essentiality of these PAMs and explore novel antipsychotics hits, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking, pharmacophore modeling, and molecular dynamics (MD) were performed on 33 imidazo [1,2-a]-pyridines. The constructed 3D-QSAR models exhibited good predictive abilities. The dockings results and MD simulations demonstrated that hydrogen bonds, π-π stackings, and hydrophobic interactions play essential roles in the binding of these novel PAMs in the GABAAR binding pocket. Four hit compounds (DS01-04) were then screened out by the combination of the constructed models and computations, including the pharmacophore model, Topomer Search, molecular dockings, ADME/T predictions, and MD simulations. The compounds DS03 and DS04, with higher docking scores and better predicted activities, were also found to be relatively stable in the binding pocket by MD simulations. These results might provide a significant theoretical direction or information for the rational design and development of novel α1-GABAAR PAMs with antipsychotic activities.

Structural determinants and regulation of spontaneous activity in GABAA receptors.

GABAA receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for ß3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the ß3 subunit's extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by ß3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABAA receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABAA receptor gating.