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Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABA(A) receptors and modulating their function. The structural mechanisms by which BZD binding is transd...
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... exert their effects on the CNS by binding to the GABA A R and allosterically modulating GABA-induced current (I GABA ) responses. The BZD binding site is located on the extracellular surface of the GABA A R, and is formed by residues located in at least six noncontiguous regions at the α/γ interface historically designated Loops A-F (Fig.1A) (reviewed in (Sigel, 2002)). ...
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... BZD binding site is located on the extracellular surface of the GABA A R, and is formed by residues located in at least six noncontiguous regions at the α/γ interface historically designated Loops A-F (Fig.1A) (reviewed in (Sigel, 2002)). This site binds a large selection of structurally diverse ligands (Fig.1B), including agonists that potentiate GABA-mediated Cl -current (I GABA ) (positive modulators such as flurazepam (FZM) and zolpidem (ZPM)), inverse agonists that inhibit I GABA (negative modulators such as DMCM), and antagonists that bind at the BZD site but have no effect on I GABA (zero modulators such as Ro15-1788). ...
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... the identified γ 2 subunit residues were not critical for inhibition of I GABA by the BZD-site inverse agonist DMCM ( Boileau and Czajkowski, 1999), suggesting negative allosteric modulation is governed by structural elements distinct from that of positive allosteric modulation. In the γ 2 subunit, a stretch of ~20 residues called Loop F links the BZD binding site to the beginning of β-strand 9 near the transmembrane channel gating domain ( Fig.1A) and thus, is in an ideal position to transduce BZD binding site movements to movements near the channel domain. ...
Citations
... Benzodiazepines bind to the GABA A R at allosteric sites and enhance the GABA A R current by increasing chloride conductance. Benzodiazepines binding to the synaptic GABA A R induce a conformational change for which GABA has a higher affinity, thereby increasing the frequency of chloride channel opening [157][158][159]. Benzodiazepines are sometimes used to treat specific symptoms associated with depression, such as anxiety and insomnia. ...
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). Most GABAergic neurons synthesize GABA from glutamate and release it in the synaptic cleft in the CNS. However, astrocytes can also synthesize and release GABA, activating GABA receptors in the neighboring neurons in physiological and pathological conditions. As the primary homeostatic glial cells in the brain, astrocytes play a crucial role in regulating GABA homeostasis and synaptic neurotransmission. Accumulating evidence demonstrates that astrocytic GABA dysregulation is implicated in psychiatric disorders, including alcohol use disorder (AUD) and major depressive disorder (MDD), the most prevalent co-occurring psychiatric disorders. Several current medications and emerging pharmacological agents targeting GABA levels are in clinical trials for treating AUD and MDD. This review offers a concise summary of the role of astrocytic GABA regulation in AUD and MDD. We also provide an overview of the current understanding and areas of debate regarding the mechanisms by which astrocytes regulate GABA in the CNS and their potential significance in the molecular basis of AUD and MDD, paving the way toward future research directions and potential therapeutic target areas within this field.
... The presence of GABA is necessary for benzodiazepine response. Binding of benzodiazepines to the synaptic GABA A receptor locks the receptor into a conformation for which GABA has much higher affinity, thus increasing the frequency of the chloride channel opening, with minimal effect on the duration of bursts [170,171]. Barbiturates, on the other hand, bind in the presence of GABA to both synaptic and extrasynaptic GABA A receptors and increase the duration of chloride channel opening without altering the frequency of bursts [169,171]. Only at high doses can barbiturates directly stimulate GABA A receptors in the absence of GABA [172]. ...
The pathophysiology of major depressive disorder (MDD) is thought to result from impaired connectivity between key brain networks. Gamma-aminobutyric acid (GABA) is the key inhibitory neurotransmitter in the brain, working primarily via GABA A receptors, with an important role in virtually all physiologic functions in the brain. Some neuroactive steroids (NASs) are positive allosteric modulators (PAMs) of GABA A receptors and potentiate phasic and tonic inhibitory responses via activation of synaptic and extrasynaptic GABA A receptors, respectively. This review first discusses preclinical and clinical data that support the association of depression with diverse defects in the GABAergic system of neurotransmission. Decreased levels of GABA and NASs have been observed in adults with depression compared with healthy controls, while treatment with antidepressants normalized the altered levels of GABA and NASs. Second, as there has been intense interest in treatment approaches for depression that target dysregulated GABAergic neurotransmission, we discuss NASs approved or currently in clinical development for the treatment of depression. Brexanolone, an intravenous NAS and a GABA A receptor PAM, is approved by the U.S. Food and Drug Administration for the treatment of postpartum depression (PPD) in patients 15 years and older. Other NASs include zuranolone, an investigational oral GABA A receptor PAM, and PH10, which acts on nasal chemosensory receptors; clinical data to date have shown improvement in depressive symptoms with these investigational NASs in adults with MDD or PPD. Finally, the review discusses how NAS GABA A receptor PAMs may potentially address the unmet need for novel and effective treatments with rapid and sustained antidepressant effects in patients with MDD.
... The γ subunit β8-β9 loop extends along the bottom outer edge of the BZD binding pocket to the intersubunit region near the ECD/TMD interface, where it can interact with the M2-M3 linker, Cys-loop, and β1-β2 loop in the neighboring α subunit, a region known to be important in channel activation [115,116] (Figure 2). This loop has been implicated in both the transduction of the modulatory effects and the binding of BZDs [57,60,[117][118][119]. The homologous loop in the α subunit has also been implicated in gating by GABA [120] and was observed to undergo relatively large motions upon the activation of the prokaryotic homologue GLIC [121]. ...
Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs that target GABAA receptors (GABAARs) to tune inhibitory synaptic signaling throughout the central nervous system. Despite knowing their molecular target for over 40 years, we still do not fully understand the mechanism of modulation at the level of the channel protein. Nonetheless, functional studies, together with recent cryo-EM structures of GABAA(α1)2(βX)2(γ2)1 receptors in complex with BZDs, provide a wealth of information to aid in addressing this gap in knowledge. Here, mechanistic interpretations of functional and structural evidence for the action of BZDs at GABAA(α1)2(βX)2(γ2)1 receptors are reviewed. The goal is not to describe each of the many studies that are relevant to this discussion nor to dissect in detail all the effects of individual mutations or perturbations but rather to highlight general mechanistic principles in the context of recent structural information.
... Additionally, this study demonstrated γ1-containing receptors had a slower rise in the amplitude of inhibitory current, suggesting that the receptors were not as concentrated at synapses as γ2-containing receptors. The γ subunit composition of GABA A Rs also has important implications for the modulation of GABA A Rs by benzodiazepines, as γ1 subunit-containing receptors are less responsive to benzodiazepines than γ2containing receptors (Hanson and Czajkowski, 2008;Keramidas and Harrison, 2010). ...
Inhibitory signaling in the brain organizes the neural circuits that orchestrate how living creatures interact with the world around them and how they build representations of objects and ideas. Without tight control at multiple points of cellular engagement, the brain’s inhibitory systems would run down and the ability to extract meaningful information from excitatory events would be lost leaving behind a system vulnerable to seizures and to cognitive decline. In this review, we will cover many of the salient features that have emerged regarding the dynamic regulation of inhibitory signaling seen through the lens of cell biology with an emphasis on the major building blocks, the ligand-gated ion channel receptors that are the first transduction point when the neurotransmitter GABA is released into the synapse. Epilepsy association will be used to indicate importance of key proteins and their pathways to brain function and to introduce novel areas for therapeutic intervention.
... Klasik olarak, benzodiazepinler GABAA reseptörü/kanalındaki benzodiazepin bağlama bölgesi aracılığıyla etki ederler ve klorür akışının artmasına ve nöronal membran potansiyelinin ve uyarılabilirliğinin (eksitabilite) azalmasına neden olurlar [7]. Benzodiazepinlerin GABAA reseptörü üzerindeki bağlanma yeri iyi bilinmesine rağmen [8], bu ilaç gruplarının antikonvülzan etki mekanizmaları henüz aydınlatılamamıştır. Nitrik oksit (NO), bir enzim ailesi olan nitrik oksit sentaz (NOS) tarafından L-arginin'den sentezlenen ve guanilil siklazı güçlü bir şekilde uyaran, ikinci haberci cGMP seviyelerinin artmasına neden olan ve serbest radikale dönüşebilen bir gazdır. ...
Bu çalışmanın amacı, sıçanlarda pentilentetrazol (PTZ) ile oluşturulan nöbetlerde diazepamın antikonvulzif etkinliğinde nitrik oksit sentaz (NOS) ve beyin kaynaklı nörotrofik faktör (BDNF) üzerine etkisini araştırmaktır. Çalışmada 24 adet erkek Wistar Albino sıçan kullanılmıştır. Sıçanlar kontrol grubu, salin (1 mL kg-1) + PTZ, diazepam (0,5 mg kg-1) + PTZ ve diazepam (2 mg kg-1)+PTZ grubu olarak 4 gruba ayrılmıştır (n=6). İntraperitoneal ilaç uygulamalarından 24 saat sonra biyokimyasal incelemeler için tüm sıçanların beyin dokuları çıkarılıp, korteks ve hipokampüs bölgeleri ayrılmıştır. Korteks ve hipokampüsteki NOS ve BDNF düzeyleri enzim bağlı immünosorbent analizi (ELISA) kitleri kullanılarak ölçülmüştür. Diazepam, salin+PTZ uygulanan grubuna göre istatistiksel açıdan anlamlı bir şekilde nöbet evresini azalttı ve ilk miyoklonik jerk (İMJ) süresini uzattı (p
... γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system 1 and mediates its effect via the ionotropic GABAA receptors (GABAARs) and the metabotropic GABAB receptors. 2 The GABAARs are clinically employed targets for numerous drugs including anesthetics 3 , barbiturates 4 and benzodiazepines 5 for their sedative, anxiolytic and anticonvulsant effects. GABAARs form transmembrane heteropentameric complexes from at least 19 different subunits including α1-6, β1-3, γ1-3, ρ1-3, δ, θ, π and . ...
... The reaction mixture was refluxed for 23 hours. The crude product was recrystallized from (4 (5 General Suzuki-Miyaura cross-coupling procedure: ...
... (9). Obtained from cyclopentanecarbaldehyde (1.0 mL, 9.3 mmol), NH4 Cl, (0.50 g, 9.3 mmol), tert-butyl isocyanide (1.3 mL, 11.5 mmol) and 2-amino-5-bromopyridine (1.61 g, 9.3 mmol) using the general GBB procedure.The reaction mixture was refluxed for 46 hours. The crude product was recrystallized from (MeCN : H 2 O) (3 : 1), which furnished the product as large, slightly grey crystals (2.10 g, 67% yield): mp 143°C. ...
div>A SAR study of the delta-selective positive modulators DS2 was performed to assist the quest for the binding site. The modulatory effect was measured using a fluorometric inaging plate reader (FLIPR) membrane potential (FMP) functional assay. Specific positions in the structural scaffold of DS2 was found to severly affect the pharmacological profile.
Analogs superior to DS2 were identified displaying higher potency and selectivity for the alfa4beta1delta over alfa4beta1gamma.
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... Well-studied modulators, such as flurazepam and zolpidem, have EC 50 s in the nanomolar range when co-applied with GABA EC 2-5 , being 270 nM and 340 nM, respectively. 37 The PAM tested here, (+)-2, appears to be less potent with an EC 50 of 110 μΜ when co-applied with GABA EC 5 , as shown in Figure 3B and Table S3. Having established that pyrroloindoline (+)-2 acts as a PAM on the α1β2γ2 GABA A receptor, further potentiation experiments used the GABA EC 10-15 instead of EC 50 . ...
γ-Aminobutyric acid type A (GABA_A) receptors are key mediators of central inhibitory neurotransmission and have been implicated in several disorders of the central nervous system. Some positive allosteric modulators (PAMs) of this receptor provide great therapeutic benefits to patients. However, adverse effects remain a challenge. Selective targeting of GABA_A receptors could mitigate this problem. Here, we describe the synthesis and functional evaluation of a novel series of pyrroloin-dolines that display significant modulation of the GABA_A receptor, acting as PAMs. We found that halogen incorporation at the C5 position greatly increased the PAM potency relative to the parent ligand, while substitutions at other positions generally decreased potency. Mutagenesis studies suggest that the binding site lies at the top of the transmembrane domain.
... The most commonly accepted notion states that BZDs increase GABA affinity by inducing a conformational change within the GABAA receptor [7]. A newer theory posits that BZDs cause receptor equilibrium to shift from the closed state to the opened state, indirectly increasing GABA binding affinity [29]. ...
... When the conformation does change, GABA can bind more easily and induce larger inhibitory currents than normal. In support of this hypothesis, one case-study found that specific residues within the transmembrane domain were required to allow subunit-receptor gating [29]. A chimeric study expanded on this theory and found that BZDs changed the γ2 subunit's conformation within the transmembrane domain. ...
... Triazolo-benzodiazepines analogues are a key structural motif in numerous therapeutics that have sedative, muscle relaxant, and antitumor activities [8,9]. Alprazolam, adinazolam and estazolam are commercially available chemical drugs based on triazolo-benzodiazepine scaffold that widely used as anxiolytic and sedative agents [10,11,12,13]. Some triazolo-benzodiazepine derivatives have been reported to be weakly bound to the benzodiazepine receptor and prevent serine protease [14,15]. ...
In this study, we have selected a series of a new family of molecules bearing Triazolo-benzodiazepines, an eleven membered heterocyclic ring has been studied for antidepression activity. Docking studies suggested that all the eleven ligands interacted well within active site of Drosophila melanogaster dopamine transporter (dDAT) (PDB ID: 4M48). Most ligands formed H-bond with amino acid Phe43, Asp46, Asp475, Tyr123, Ser421 and/or Gln316 and also exhibited Pi and Pi-Pi interactions with amino acid residues Tyr124, Phe319, Phe43, Phe325, Ala479 and Val120. In silico ADME evaluations of compounds showed more than 96% intestinal absorption for all compounds. During in vitro Toxicity properties prediction, the Triazolo-benzodiazepines derivatives: M1, M2, M3 and M11 showed less toxicity than the other studied molecules against algae, for daphnia the molecules M1, M2, M3, M8, M10 and M11 showed less toxicity than the reference molecule (Nortriptyline).
... Loop C is important for ligand binding because it has more mobility than the other loops 35 and may affect benzodiazepine ligand selectivity. 36 Previous studies found that the α6(Asn204) and α4(Ile203) residues (both homologous to human α1(Ser206)) were important for distinguishing the binding of negative benzodiazepines. 19 Ser206 also physically interacts with diazepam in α1, α2 ...
Many benzodiazepines are positive allosteric modulators (PAMs) of GABAA receptors that cause sedation, hypnosis, and anxiolysis. Benzodiazepines bind GABAA receptors at the extracellular interface of the α and γ subunits. Within the α subunit, the benzodiazepine binding site is defined by three highly conserved structural loops, loops A‐C. Although previous mutagenesis studies have identified His102 in Loop A as important for benzodiazepine modulation of GABAA receptors, the functional roles of many of the other conserved residues in loops A‐C remain incompletely understood. In this study, we made single mutations in loops A‐C of the benzodiazepine binding‐site across all six α subunits. We used whole‐cell patch clamp recording to measure the functional effects of these mutations on midazolam potentiation. The results showed that mutating the threonine in loop B and serine in loop C (Thr163 and S206 in human α1) did not abolish the receptors’ responsiveness to midazolam, as the α1(H102R) mutation did. The loop C mutations exhibited a novel array of α‐isoform specific effects on midazolam potentiation. The α3(S230I) and α5(S209I) mutations had the largest effect on midazolam potentiation, increasing the efficacy of midazolam. Novel benzodiazepines targeting loop C may represent a future direction for designing new drugs that specifically alter the activity of α3‐ and α5‐containing GABAA receptors.
