ORCID Profile
0000-0002-5455-8013
Current Organisations
IT University of Copenhagen
,
Københavns Universitet
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Publisher: Springer Science and Business Media LLC
Date: 05-03-2015
Publisher: SAGE Publications
Date: 17-11-2017
Abstract: Brain ischemia triggers excitotoxicity and cell death, yet no neuroprotective drugs have made it to the clinic. While enhancing GABAergic signaling to counterbalance excitotoxicity has shown promise in animal models, clinical studies have failed. Blockade of GABA transporters (GATs) offers an indirect approach to increase GABA inhibition to lower the excitation threshold of neurons. Among the GATs, GAT1 is known to promote neuroprotection, while the protective role of the extrasynaptic transporters GAT3 and BGT1 is elusive. A focal lesion was induced in the motor cortex in two to four-month-old C57BL/6 J male mice by photothrombosis. The GAT1 inhibitor, tiagabine (1 and 10 mg/kg), the GAT2/3 inhibitor, ( S)-SNAP-5114 (5 and 30 mg/kg) and the GAT1/BGT1 inhibitor, EF-1502 (1 and 10 mg/kg) were given i.p. 1 and 6 h post-stroke to assess their impact on infarct volume and motor performance seven days post-stroke. One mg/kg tiagabine improved motor performance, while 10 mg/kg tiagabine, ( S)-SNAP-5114 and EF-1502 had no effect. None of the compounds affected infarct volume. Interestingly, treatment with tiagabine induced seizures and ( S)-SNAP-5114 led to increased mortality. Although we show that tiagabine can promote protection, our findings indicate that caution should be had when using GAT1 and GAT3 inhibitors for conditions of brain ischemia.
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S0028-3908(02)00041-2
Abstract: The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known H(3) agonists, while all agonists tested displayed increased potency at isoform 2 relative to isoform 1. Histamine, N(alpha)-methylhistamine, and R(-) and S(+)-alpha-methylhistamine are 16-23-fold more potent, while immepip and imetit are three to fivefold more potent. Antagonist experiments revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor activity, and identified a small number of antipsychotics that possess significant antagonist activity.
Publisher: Elsevier
Date: 2010
Publisher: Oxford University Press (OUP)
Date: 20-05-2023
Abstract: Narcolepsy type 1 (NT1) is a neurological disorder caused by disruption of hypocretin (HCRT or orexin) neurotransmission leading to fragmented sleep/wake states, excessive daytime sleepiness, and cataplexy (abrupt muscle atonia during wakefulness). Electroencephalography and electromyography (EEG/EMG) monitoring is the gold standard to assess NT1 phenotypical features in both humans and mice. Here, we evaluated the digital ventilated home-cage (DVC®) activity system as an alternative to detect NT1 features in two NT1 mouse models: the genetic HCRT-knockout (-KO) model, and the inducible HCRT neuron-ablation hcrt-tTA TetO-DTA (DTA) model, including both sexes. NT1 mice exhibited an altered dark phase activity profile and increased state transitions, compared to the wild-type (WT) phenotype. An inability to sustain activity periods & min represented a robust activity-based NT1 biomarker. These features were observable within the first weeks of HCRT neuron degeneration in DTA mice. We also created a nest-identification algorithm to differentiate between inactivity and activity, inside and outside the nest as a sleep and wake proxy, respectively, showing significant correlations with EEG/EMG-assessed sleep/wake behavior. Lastly, we tested the sensitivity of the activity system to detect behavioral changes in response to interventions such as repeated saline injection and chocolate. Surprisingly, daily consecutive saline injections significantly reduced activity and increased nest time of HCRT-WT mice. Chocolate increased total activity in all mice, and increased the frequency of short out-of-nest inactivity episodes in HCRT-KO mice. We conclude that the DVC® system provides a useful tool for non-invasive monitoring of NT1 phenotypical features, and has the potential to monitor drug effects in NT1 mice.
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.BCP.2011.09.002
Abstract: Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA(A) receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA(A) receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2'-methoxy-6-methylflavone (3-OH-2'MeO6MF) potentiates GABA-induced currents at recombinant α1/2β2, α1/2/4/6β1-3γ2L but not α3/5β1-3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC(50) values between 38 and 106 μM) and occurred in a flumazenil-insensitive manner. 3-OH-2'MeO6MF displayed preference for β2/3- over β1-containing receptors with the highest efficacy observed at α2β2/3γ2L, displaying a 4-11-fold increase in efficacy over α2β1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2'MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4β2/3δ receptors expressed in oocytes. The affinity of 3-OH-2'MeO6MF for α4β2/3δ receptors (EC(50) values between 1.4 and 2.5 μM) was 10-fold higher than at α4β1δ GABA(A) receptors. 3-OH-2'MeO6MF acted as a full agonist at α4β2/3δ (105% of the maximal GABA response) but as a partial agonist at α4β1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2'MeO6MF (1-100 mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2'MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2β2/3γ2L and direct activation of α4β2/3δ GABA(A) receptor subtypes.
Publisher: American Chemical Society (ACS)
Date: 22-05-2019
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 02-12-2004
Abstract: One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRC6A. To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with alpha-amino acids and was employed to rationally select potential ligands. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/5.24 and identification of L-alpha-amino acids as agonists. The most active agonists were basic L-alpha-amino acids, L-Arg, L-Lys, and L-ornithine, suggesting that these may function as endogenous signaling molecules. Measurement of intracellular calcium in tsA cells expressing h6A/5.24 allowed determination of EC50 values, which confirmed the agonist preferences observed in oocytes. Cloning, cell surface expression and deorphanization of the mouse ortholog further reinforces the assignment of the agonist preferences of hGPRC6A. This study demonstrates the utility of a chimeric receptor approach in combination with molecular modeling, for elucidating agonist interaction with GPRC6A, a novel family C G-protein-coupled receptor.
Publisher: Wiley
Date: 17-05-2021
DOI: 10.1111/EJN.15267
Abstract: γ‐Aminobutyric acid (GABA) acting through heteropentameric GABA A receptors plays a pivotal role in the sleep‐promoting circuitry. Whereas the role of the different GABA A receptor α‐subunits in sleep regulation and in mediating the effect of benzodiazepines for treatment of insomnia is well‐described, the β‐subunits are less studied. Here we report the first study characterizing sleep in mice lacking the GABA A receptor β 1 ‐subunit (β 1 −/− mice). We show that β 1 −/− mice have a distinct and abnormal sleep phenotype characterized by increased delta power in non‐rapid eye movement (NREM) sleep and decreased theta activity in rapid eye movement (REM) sleep compared to β 1 +/+ mice, without any change in the overall sleep‐wake architecture. From GABA A receptor‐specific autoradiography, it is further demonstrated that functional β 1 ‐subunit‐containing GABA A receptors display the highest binding levels in the hippoc us and frontal cortex. In conclusion, this study suggests that the GABA A receptor β 1 ‐subunit does not play an important role in sleep initiation or maintenance but instead regulates the power spectrum and especially the expression of theta rhythm. This provides new knowledge on the complex role of GABA A receptor subunits in sleep regulation. In addition, β 1 −/− mice could provide a useful mouse model for future studies of the physiological role of delta and theta rhythms during sleep.
Publisher: Elsevier BV
Date: 11-2022
DOI: 10.1016/J.VASCN.2022.107226
Abstract: Ca
Publisher: Elsevier BV
Date: 05-2023
Publisher: American Chemical Society (ACS)
Date: 02-05-2022
DOI: 10.1021/ACS.JMEDCHEM.1C02177
Abstract: The Ca
Publisher: Elsevier BV
Date: 11-2011
DOI: 10.1016/J.CHEMBIOL.2011.09.012
Abstract: GPRC6A is a Family C G protein-coupled receptor recently discovered and deorphanized by our group. This study integrates chemogenomic ligand inference, homology modeling, compound synthesis, and pharmacological mechanism-of-action studies to disclose two noticeable results of methodological and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of pharmacological activity across GPCR families provides proof-of-concept for in silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors containing the chemogenomic binding sequence motif where some of the identified GPRC6A antagonists showed some activity. However, three compounds with at least ∼3-fold selectivity for GPRC6A were discovered, which present a significant step forward compared with the previously published GPRC6A antagonists, calindol and NPS 2143, which both display ∼30-fold selectivity for the calcium-sensing receptor compared to GPRC6A. The antagonists constitute novel research tools toward investigating the signaling mechanism of the GPRC6A receptor at the cellular level and serve as initial ligands for further optimization of potency and selectivity enabling future ex vivo/in vivo pharmacological studies.
Publisher: Wiley
Date: 03-2006
Publisher: Portland Press Ltd.
Date: 03-2023
DOI: 10.1042/EBC20220208
Abstract: Synaptic regulation of the primary inhibitory neurotransmitter γ-aminobutyric acid (GABA) is essential for brain function. Cerebral GABA homeostasis is tightly regulated through multiple mechanisms and is directly coupled to the metabolic collaboration between neurons and astrocytes. In this essay, we outline and discuss the fundamental roles of astrocytes in regulating synaptic GABA signaling. A major fraction of synaptic GABA is removed from the synapse by astrocytic uptake. Astrocytes utilize GABA as a metabolic substrate to support glutamine synthesis. The astrocyte-derived glutamine is subsequently transferred to neurons where it serves as the primary precursor of neuronal GABA synthesis. The flow of GABA and glutamine between neurons and astrocytes is collectively termed the GABA-glutamine cycle and is essential to sustain GABA synthesis and inhibitory signaling. In certain brain areas, astrocytes are even capable of synthesizing and releasing GABA to modulate inhibitory transmission. The majority of oxidative GABA metabolism in the brain takes place in astrocytes, which also leads to synthesis of the GABA-related metabolite γ-hydroxybutyric acid (GHB). The physiological roles of endogenous GHB remain unclear, but may be related to regulation of tonic inhibition and synaptic plasticity. Disrupted inhibitory signaling and dysfunctional astrocyte GABA handling are implicated in several diseases including epilepsy and Alzheimer’s disease. Synaptic GABA homeostasis is under astrocytic control and astrocyte GABA uptake, metabolism, and recycling may therefore serve as relevant targets to ameliorate pathological inhibitory signaling.
Publisher: American Chemical Society (ACS)
Date: 13-04-2021
Publisher: Wiley
Date: 28-06-2008
Publisher: Springer Science and Business Media LLC
Date: 03-08-2020
DOI: 10.1038/S41598-020-69908-W
Abstract: We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA transporter 1 (BGT1) (IC 50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N 1 -, exocyclic- N -, and C 4 -substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC 50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.
Publisher: Wiley
Date: 15-07-2020
DOI: 10.1111/BPH.15146
Publisher: SAGE Publications
Date: 21-11-2017
Abstract: Ischemic stroke triggers an elevation in tonic GABA inhibition that impairs the ability of the brain to form new structural and functional cortical circuits required for recovery. This stroke-induced increase in tonic inhibition is caused by impaired GABA uptake via the glial GABA transporter GAT3, highlighting GAT3 as a novel target in stroke recovery. Using a photothrombotic stroke mouse model, we show that GAT3 protein levels are decreased in peri-infarct tissue from 6 h to 42 days post-stroke. Prior studies have shown that GAT substrates can increase GAT surface expression. Therefore, we aimed to assess whether the GAT3 substrate, L-isoserine, could increase post-stroke functional recovery. L-Isoserine (38 µM or 380 µM) administered directly into the infarct from day 5 to 32 post-stroke, significantly increased motor performance in the grid-walking and cylinder tasks in a concentration-dependent manner, without affecting infarct volumes. Additionally, L-isoserine induced a lasting increase in GAT3 expression in peri-infarct regions accompanied by a small decrease in GFAP expression. This study is the first to show that a GAT3 substrate can increase GAT3 expression and functional recovery after focal ischemic stroke following a delayed long-term treatment. We propose that enhancing GAT3-mediated uptake d ens tonic inhibition and promotes functional recovery after stroke.
Publisher: Cold Spring Harbor Laboratory
Date: 12-03-2022
DOI: 10.1101/2022.03.10.483849
Abstract: Ca 2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca 2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented GHB ligands as the first small molecules selectively targeting the CaMKIIα hub, a domain primarily responsible for holoenzyme oligomerisation, with an emerging functional role. Here, we report that the GHB ligand, HOCPCA, improves sensorimotor function after experimental stroke in mice when administered at clinically relevant time and in combination with alteplase. We observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated the ischemia-specific expression of a constitutively active CaMKII kinase fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. HOCPCA’s selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5MD00442J
Abstract: Within the continuous quest for the discovery of pharmacologically interesting compounds, the development of new and superior drug screening assays is desired.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 06-2009
Abstract: A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for ex le, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing L-alpha-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the in idual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for ex le, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.
Publisher: SAGE Publications
Date: 07-04-2023
DOI: 10.1177/0271678X231167920
Abstract: Ca 2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca 2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippoc al neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA’s effects on d ening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA’s selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 14-05-2021
Publisher: Elsevier BV
Date: 06-2004
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-09-2013
Abstract: The GPRC6A receptor is a recently "deorphanized" class C G protein-coupled receptor. We and others have shown that this receptor is coactivated by basic l-α-amino acids and alent cations, whereas other groups have also suggested osteocalcin and testosterone to be agonists. Likewise, the GPRC6A receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort to establish fully the signaling properties of the receptor, we tested representatives of four previously reported GPRC6A agonist classes for activity in the Gq, Gs, Gi, and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic l-α-amino acids and alent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling neither could we detect agonistic activity of testosterone and osteocalcin. Generation of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in high-throughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/J.NEUROPHARM.2019.05.021
Abstract: ɣ-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Publisher: Springer International Publishing
Date: 2017
DOI: 10.1007/978-3-319-55769-4_14
Abstract: Inactivation of GABA-mediated neurotransmission is achieved by high-affinity transporters located at both GABAergic neurons and the surrounding astrocytes. Early studies of the pharmacological properties of neuronal and glial GABA transporters suggested that different types of transporters might be expressed in the two cell types, and such a scenario was confirmed by the cloning of four distinctly different GABA transporters from a number of different species. These GABA-transport entities have been extensively characterized using a large number of GABA analogues of restricted conformation, and several of these compounds have been shown to exhibit pronounced anticonvulsant activity in a variety of animal seizure models. As proof of concept of the validity of this drug development approach, one GABA-transport inhibitor, tiagabine, has been developed as a clinically active antiepileptic drug. This review provides a detailed account of efforts to design new subtype-selective GABA-transport inhibitors aiming at identifying novel antiepileptic drug candidates.
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.BCP.2013.10.028
Abstract: γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.
Publisher: American Chemical Society (ACS)
Date: 22-05-2019
DOI: 10.1021/ACS.JMEDCHEM.9B00026
Abstract: Given the heterogeneity within the γ-aminobutyric acid (GABA) receptor and transporter families, a detailed insight into the pharmacology is still relatively sparse. To enable studies of the physiological roles governed by specific receptor and transporter subtypes, a series of GABA analogues comprising five-membered nitrogen- and sulfur-containing heterocycles as amine bioisosteres were synthesized and pharmacologically characterized at native and selected recombinant GABA
Publisher: American Chemical Society (ACS)
Date: 17-11-2016
DOI: 10.1021/ACSCHEMNEURO.6B00335
Abstract: γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the
Publisher: Elsevier BV
Date: 02-2020
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.NEUINT.2016.09.002
Abstract: GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version (
Publisher: Wiley
Date: 03-2009
Publisher: Cold Spring Harbor Laboratory
Date: 14-03-2022
DOI: 10.1101/2022.03.13.484119
Abstract: Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection. Ameen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection. Identification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity. The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons. These pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death. Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection. (A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKIIα and CaMKIIβ, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotection Proteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.
Publisher: Wiley
Date: 24-07-2020
DOI: 10.1002/JLCR.3870
Publisher: MDPI AG
Date: 22-04-2022
DOI: 10.3390/PHARMACEUTICS14050919
Abstract: Clove oil (CO), an essential oil of Syzygium aromaticum, has been reported as an anesthetic for many fish species. However, its insoluble properties require a suitable delivery system for its application. In the present study, nanoformulations of CO as a nanoemulsion (CO-NE), a self-microemulsifying drug-delivery system (CO-SMEDDS), and a self-nanoemulsifying drug-delivery system (CO-SNEDDS) were prepared for delivering CO. Zebrafish were used as a fish model to investigate oil pathways. The result shows fluorescence spots of fluorescence-labeled CO accumulate on the gills, skin, and brain. All CO nanoformulations significantly increased penetration flux compared to CO ethanolic solution. Investigation of the anesthetic mechanism of action using a rat brain γ-aminobutyric acid subtype A (GABAA) receptor-binding test demonstrates that CO and its major compound, eugenol, modulate [3H]muscimol binding. CO-NE exhibited a concentration-dependent binding activity with an EC50 value of 175 µg/mL, significantly higher than CO solution in dimethyl sulfoxide. In conclusion, CO enters the fish through the skin and gills. The anesthetic mechanism of action of CO is based on modulation of [3H] muscimol binding to GABAA receptors. Among three nanoformulations tested, CO-NE is the most effective at increasing permeability and enhancing the receptor-binding activity of the oil.
Publisher: Wiley
Date: 31-01-2019
DOI: 10.1111/JNC.14649
Abstract: γ‐Hydroxybutyric acid ( GHB ) is an endogenous compound proposed to act as a neurotransmitter. Na + ‐dependent, high‐affinity GHB transport has long been considered important evidence supporting this hypothesis. However, the molecular identity of such a high‐affinity transporter remains unknown. In this study, we sought to identify and characterize GHB synaptic transport through a series of studies using both native and recombinant systems with the ultimate aim of providing evidence to clarify the proposed role of GHB as a neurotransmitter in the mammalian brain. Native [ 3 H] GHB transport was studied in isolated rat brain synaptosomes and compared to synaptic membranes. As a targeted approach, GHB was also screened against a panel of Na + ‐dependent SLC 6 neurotransmitter transporters recombinantly expressed in Xenopus laevis oocytes or tsA201 cells. Finally, the low‐affinity GHB transporters, MCT 1/2 and SMCT 1, were probed as GHB transporters in L‐[ 14 C]lactate uptake assays in synaptosomes. We found no evidence of high‐affinity [ 3 H] GHB transport in purified rat brain cortical or striatal synaptosomes or at any of the 11 SLC 6 transporters tested. Instead, our results indicate the binding of [ 3 H] GHB to an unidentified membrane component, distinct from any of the known GHB targets. In accordance with others, we found that GHB and the analog 3‐hydroxycyclopent‐1‐enecarboxylic acid (HOCPCA) can, in millimolar concentrations, inhibit L‐[ 14 C]lactate uptake at MCT 1 and/or MCT 2 and that this also can occur in synaptosomes. In conclusion, through a variety of in vitro pharmacological studies, we were unsuccessful in identifying a specific synaptic high‐affinity transporter for GHB . Our findings emphasize the need to reevaluate GHB 's role as a potential neurotransmitter. This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at cos.io/our-services/open-science-badges/ . image
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 09-08-2010
Abstract: γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippoc us and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([(3)H]NCS-382) binding sites. Using a (125)I-labeled photoaffinity derivative of the new GHB ligand, we have performed denaturing protein electrophoresis and detected one major protein band with an apparent mass of 50 kDa from cortical and hippoc al membranes. [(125)I]BnOPh-GHB is the first reported (125)I-labeled GHB radioligand and is a useful tool for in vitro studies of the specific high-affinity GHB binding sites. The related photoaffinity linker [(125)I]4-hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate can be used as a probe for isolation of the elusive GHB binding protein.
Publisher: Wiley
Date: 07-04-2023
DOI: 10.1111/JNC.15814
Abstract: Ca 2+ /calmodulin‐dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable 13 C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U‐ 13 C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2‐ 13 C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U‐ 13 C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U‐ 13 C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain. image
Publisher: American Chemical Society (ACS)
Date: 27-02-2013
DOI: 10.1021/JM301872X
Abstract: β-Amino acids sharing a lipophilic diaromatic side chain were synthesized and characterized pharmacologically on mouse GABA transporter subtypes mGAT1-4. The parent amino acids were also characterized. Compounds 13a, 13b, and 17b displayed more than 6-fold selectivity for mGAT2 over mGAT1. Compound 17b displayed anticonvulsive properties inferring a role of mGAT2 in epileptic disorders. These results provide new neuropharmacological tools and a strategy for designing subtype selective GABA transport inhibitors.
Publisher: Springer Science and Business Media LLC
Date: 23-05-2014
DOI: 10.1007/S11064-014-1336-9
Abstract: The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [(3)H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC50 value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.
Publisher: Elsevier BV
Date: 2003
DOI: 10.1016/S0223-5234(02)00003-X
Abstract: A general, improved procedure for rapid synthesis of philanthotoxin analogues, a pharmacologically important class of polyamine conjugates, is described. The solution-phase procedure is illustrated by gram-scale synthesis of philanthotoxins PhTX-343 and PhTX-12. Selectively protected polyamines are coupled to N(alpha)-Fmoc-protected amino acid pentafluorophenyl esters. After removal of the N(alpha)-Fmoc group, the amine is coupled with carboxylic acid pentafluorophenyl esters. Deprotection followed by a rapid and efficient purification by vacuum liquid chromatography on octadecylsilyl silica (RP-18 phase) gave the philanthotoxin analogues in 74-78% overall yield.
Publisher: Springer Science and Business Media LLC
Date: 06-07-2017
DOI: 10.1038/S41598-017-05058-W
Abstract: Virtual screening offers an efficient alternative to high-throughput screening in the identification of pharmacological tools and lead compounds. Virtual screening is typically based on the matching of target structures or ligand pharmacophores to commercial or in-house compound catalogues. This study provides the first proof-of-concept for our recently reported method where pharmacophores are instead constructed based on the inference of residue-ligand fragments from crystal structures. We demonstrate its unique utility for G protein-coupled receptors, which represent the largest families of human membrane proteins and drug targets. We identified five neutral antagonists and one inverse agonist for the histamine H 3 receptor with potencies of 0.7–8.5 μM in a recombinant receptor cell-based inositol phosphate accumulation assay and validated their activity using a radioligand competition binding assay. H 3 receptor antagonism is of large therapeutic value and our ligands could serve as starting points for further lead optimisation. The six ligands exhibit four chemical scaffolds, whereof three have high novelty in comparison to the known H 3 receptor ligands in the ChEMBL database. The complete pharmacophore fragment library is freely available through the GPCR database, GPCRdb, allowing the successful application herein to be repeated for most of the 285 class A GPCR targets. The method could also easily be adapted to other protein families.
Publisher: Wiley
Date: 15-05-2010
DOI: 10.1002/JLCR.1767
Abstract: Addresses by the three of the four Wiley Young Scientist Award winners are highlighted. Copyright © 2010 John Wiley & Sons, Ltd.
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.EJMECH.2017.06.024
Abstract: The α
Publisher: American Chemical Society (ACS)
Date: 19-10-2017
DOI: 10.1021/ACS.JMEDCHEM.7B00924
Abstract: N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors.
Publisher: Springer Science and Business Media LLC
Date: 07-2023
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.EJPHAR.2006.02.015
Abstract: The goldfish odorant receptor 5.24 is a member of family C of G protein-coupled receptors and is closely related to the human receptor GPRC6A. Receptor 5.24 has previously been shown to have binding affinity for L-alpha-amino acids, especially the basic amino acids arginine and lysine. Here we report the agonist activities of the 20 proteinogenic L-alpha-amino acids, and L-ornithine and L-citrulline, measured in an intracellular calcium release assay in mammalian tsA cells. The results show that receptor 5.24 is broadly activated by 19 of the tested L-alpha-amino acids and displays a preference for basic amino acids.
Publisher: American Chemical Society (ACS)
Date: 13-11-2017
DOI: 10.1021/ACSCHEMNEURO.7B00351
Abstract: The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABA
Publisher: Springer Science and Business Media LLC
Date: 20-06-2012
DOI: 10.1007/S00726-012-1341-8
Abstract: GPRC6A is a seven-transmembrane receptor activated by a wide range of L-α-amino acids, most potently by L-arginine and other basic amino acids. The receptor is broadly expressed, but its exact physiological role remains to be elucidated. It is well established that L-arginine stimulates insulin secretion therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by L-arginine did not stimulate insulin secretion. We also investigated central metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in L-arginine-induced insulin release and glucose metabolism under normal physiological conditions.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2023
DOI: 10.1007/S00044-023-03126-7
Abstract: The GABA transporter 3 (GAT3) is a member of the GABA transporter (GAT) family proposed to have a role in regulating tonic inhibition. The GAT3-preferring substrate ( S )-isoserine has shown beneficial effects in a mouse model of stroke accompanied by an increased GAT3 expression, indicating a molecular mechanism mediated by GAT3. However, ( S )-isoserine is not ideally suited for in vivo studies due to a lack of selectivity and brain permeability. To elucidate the structural determinants of ( S )-isoserine for GAT3 inhibition, and to optimize and inform further ligand development, we here present the design, synthesis and pharmacological evaluation of a series of conformationally constrained isoserine analogs with defined stereochemistry. Using [ 3 H]GABA uptake assays at recombinant human GAT3, we identified the azetidine and pyrrolidine analogs (2 S ,2´ S )- 6 and (2 S ,2´ S )- 7 as the most potent inhibitors. To further elaborate on the selectivity profile both compounds were tested at all GATs, the taurine transporter (TauT) and GABA A receptors. Although (2 S ,2´ S )- 6 and (2 S ,2´ S )- 7 are comparable to ( S )-isoserine with respect to potency, the selectivity vs. the taurine transporter was significantly improved (at least 6 and 53 times more activity at hGAT3, respectively). A subsequent comprehensive structure-activity study showed that different connectivity approaches, stereochemical variations, simple or larger α- and N -substituents, and even minor size enlargement of the heterocyclic ring all abrogated GAT3 inhibition, indicating very strict stereochemical and size requirements. The observed structure activity relationships may guide future ligand optimization and the novel ligands ((2 S ,2´ S )- 6 and (2 S ,2´ S )- 7 ) can serve as valuable tools to validate the proposed GAT3-mediated effect of ( S )-isoserine such as in functional recovery after stroke and thus help corroborate the relevance of targeting GAT3 and tonic inhibition in relevant brain pathologies.
Publisher: Bioscientifica
Date: 22-12-2008
DOI: 10.1677/JME-08-0149
Abstract: GPRC6A is a seven-transmembrane receptor mediating signaling by a wide range of l -α-amino acids, a signaling augmented by the alent cations Ca 2 + and Mg 2 + . GPRC6A transcripts are detected in numerous mammalian tissues, but the physiological role of the receptor is thus far elusive. Analogously to the closely related calcium-sensing receptor, GPRC6A has been proposed to function as a metabolic sensor of Ca 2 + and amino acids in bone and other tissues. In the present study, we have generated the first GPRC6A knockout mice and studied their phenotype with particular focus on bone homeostasis. The generated GPRC6A knockout mice are viable and fertile, develop normally, and exhibit no significant differences in body weight compared with wild-type littermates. Assessment of bone mineral density, histomorphometry, and bone metabolism demonstrated no significant differences between 13-week-old knockout and wild-type mice. In conclusion, our data do not support a role for GPRC6A in normal bone physiology.
Publisher: American Chemical Society (ACS)
Date: 08-11-2022
DOI: 10.1021/ACS.JMEDCHEM.2C00805
Abstract: Ca
Publisher: Proceedings of the National Academy of Sciences
Date: 02-07-2012
Abstract: γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A receptors in Xenopus laevis oocytes using the two-electrode voltage cl technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC 50 = 140 nM) over α4β(2/3)δ (EC 50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ 3 H]( E , RS )-(6,7,8,9-tetrahydro-5-hydroxy-5 H -benzocyclohept-6-ylidene)acetic acid showed a 39% reduction ( P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA A receptors and postulate a role for extrasynaptic α4δ-containing GABA A receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.
Publisher: MDPI AG
Date: 16-03-2022
DOI: 10.3390/PHARMACEUTICS14030650
Abstract: The anesthetic effect of Alpinia galanga oil (AGO) has been reported. However, knowledge of its pathway in mammals is limited. In the present study, the binding of AGO and its key compounds, methyl eugenol, 1,8-cineole, and 4-allylphenyl acetate, to gamma-aminobutyric acid type A (GABAA) receptors in rat cortical membranes, was investigated using a [3H]muscimol binding assay and an in silico modeling platform. The results showed that only AGO and methyl eugenol displayed a positive modulation at the highest concentrations, whereas 1,8-cineole and 4-allylphenyl acetate were inactive. The result of AGO correlated well to the amount of methyl eugenol in AGO. Computational docking and dynamics simulations into the GABAA receptor complex model (PDB: 6X3T) showed the stable structure of the GABAA receptor–methyl eugenol complex with the lowest binding energy of −22.16 kcal/mol. This result shows that the anesthetic activity of AGO and methyl eugenol in mammals is associated with GABAA receptor modulation. An oil-in-water nanoemulsion containing 20% w/w AGO (NE-AGO) was formulated. NE-AGO showed a significant increase in specific [3H]muscimol binding, to 179% of the control, with an EC50 of 391 µg/mL. Intracellular studies show that normal human cells are highly tolerant to AGO and the nanoemulsion, indicating that NE-AGO may be useful for human anesthesia.
Publisher: Springer Science and Business Media LLC
Date: 26-10-2023
Publisher: Proceedings of the National Academy of Sciences
Date: 30-07-2021
Abstract: GHB is a natural brain metabolite of GABA, previously reported to be neuroprotective. However, the high-affinity binding site for GHB has remained elusive for almost 40 y. We here unveil CaMKIIα, a highly important neuronal kinase, as the long-sought-after GHB high-affinity target. Via a specific interaction within the central hub domain of CaMKIIα, GHB analogs act to stabilize the hub oligomer complex. This interaction potentially explains pronounced neuroprotective effects of GHB analogs in cultured neurons exposed to a chemical insult and in mice exposed to ischemia. The postischemic treatment effects of GHB analogs underline these compounds as selective and high-affinity potential drug candidates and CaMKIIα as a relevant pharmacological target for stroke therapy.
Publisher: Elsevier BV
Date: 12-2022
DOI: 10.1016/J.BIOPHA.2022.113895
Abstract: Ca
Publisher: Elsevier BV
Date: 2017
Publisher: Springer International Publishing
Date: 2017
DOI: 10.1007/978-3-319-55769-4_7
Abstract: Imbalances in GABA-mediated tonic inhibition are involved in several pathophysiological conditions. A classical way of controlling tonic inhibition is through pharmacological intervention with extrasynaptic GABA
Publisher: Wiley
Date: 25-05-2017
DOI: 10.1111/BCPT.12778
Abstract: δ-Containing GABA
Publisher: American Chemical Society (ACS)
Date: 15-12-2021
DOI: 10.1021/ACS.JMEDCHEM.1C00290
Abstract: The 3,9-diazaspiro[5.5]undecane-based compounds
Publisher: American Chemical Society (ACS)
Date: 23-07-2015
DOI: 10.1021/ACSCHEMNEURO.5B00150
Abstract: Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-SNAP-5114, in terms of potency (low micromolar IC50 values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound 20 (5-(thiophen-2-yl)indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes.
Publisher: American Chemical Society (ACS)
Date: 14-02-2019
DOI: 10.1021/ACS.JMEDCHEM.9B00131
Abstract: Gabazine, a γ-aminobutyric acid type A (GABA
Publisher: MyJove Corporation
Date: 12-03-2019
DOI: 10.3791/58879
Abstract: In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for ex le, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity γ-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2021
DOI: 10.1101/2021.01.21.427670
Abstract: Delta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by δ subunit-containing GABA A receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α 4 β 1 δ receptors: an α 4 (+) δ (-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ 2 receptors, and two sites in the transmembrane domain (TMD) one in the α 4 (+) β 1 (-) and one in the α 4 (-) β 1 (+) interface, with the α 4 (-) β 1 (+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α 4 (+) β 1 (-) interface, either α 4 (S303L) of the α 4 (+) -side or β 1 (I289Q) of the β 1 (-) -side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patchcl electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points towards a functional selectivity of DS2 and its analogs via a surprisingly well-conserved binding pocket in the TMD. δ-Containing GABA A receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition. Yet, no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the know compound DS2, the ground is laid for design of ligands that selectively target δ-containing GABA A receptor subtypes, for better understanding of tonic inhibition, and, ultimately, for rational development of novel drugs.
Publisher: MDPI AG
Date: 29-11-2022
Abstract: Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca2+-dependent signaling pathways in various cell types throughout the body. Its neuronal isoform CaMKIIα (alpha) centrally integrates physiological but also pathological glutamate signals directly downstream of glutamate receptors and has thus emerged as a target for ischemic stroke. Previous studies provided evidence for the involvement of CaMKII activity in ischemic cell death by showing that CaMKII inhibition affords substantial neuroprotection. However, broad inhibition of this central kinase is challenging because various essential physiological processes like synaptic plasticity rely on intact CaMKII regulation. Thus, specific strategies for targeting CaMKII after ischemia are warranted which would ideally only interfere with pathological activity of CaMKII. This review highlights recent advances in the understanding of how ischemia affects CaMKII and how pathospecific pharmacological targeting of CaMKII signaling could be achieved. Specifically, we discuss direct targeting of CaMKII kinase activity with peptide inhibitors versus indirect targeting of the association (hub) domain of CaMKIIα with analogues of γ-hydroxybutyrate (GHB) as a potential way to achieve more specific pharmacological modulation of CaMKII activity after ischemia.
Publisher: Elsevier BV
Date: 03-2001
Publisher: American Chemical Society (ACS)
Date: 08-06-2022
DOI: 10.1021/ACS.ORGLETT.2C01346
Abstract: Novel γ-aminobutyric acid (GABA) analogues
Publisher: Springer Science and Business Media LLC
Date: 22-06-2020
DOI: 10.1038/S41598-020-66821-0
Abstract: Brain GABA Α receptors are ionotropic receptors belonging to the class of Cys-loop receptors and are important drug targets for the treatment of anxiety and sleep disorders. By screening a compound library (2,112 compounds) at recombinant human α 4 β 1 δ GABA Α receptors heterologously expressed in a HEK cell line, we identified a scaffold of spirocyclic compounds with nanomolar antagonist activity at GABA Α receptors. The initial screening hit 2027 (IC 50 of 1.03 μM) was used for analogue search resulting in 018 (IC 50 of 0.088 μM). 018 was most potent at α 3,4,5 -subunit containing receptors, thus showing preference for forebrain-expressed extrasynaptic receptors. Schild analysis of 018 at recombinant human α 4 β 1 δ receptors and displacement of [ 3 H]muscimol binding in rat cortical homogenate independently confirmed a competitive profile. The antagonist profile of 018 was further validated by whole-cell patch-cl electrophysiology, where kinetic studies revealed a slow dissociation rate and a shallow hill slope was observed. Membrane permeability studies showed that 2027 and 018 do not cross membranes, thus making the compounds less attractive for studying central GABA Α receptors effects, but conversely more attractive as tool compounds in relation to emerging peripheral GABA Α receptor-mediated effects of GABA e.g. in the immune system.
Publisher: Frontiers Media SA
Date: 14-09-2021
DOI: 10.3389/FCHEM.2021.736457
Abstract: The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1 S ,2 S ,5 R )-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [ 3 H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N -methylated analog ( 2 ) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (p K B value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α 1 β 2 γ 2 GABA A receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N -methylated 2 was completely devoid of activity at GABA A receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico -guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.EJPHAR.2014.06.028
Abstract: γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the in idual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.
Publisher: Wiley
Date: 03-2007
Publisher: Bentham Science Publishers Ltd.
Date: 2007
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.BCP.2012.05.017
Abstract: Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage cl technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24 nM) and α4β3δ (EC₅₀=12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16 nM EC₅₀(2)=1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA(A) receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA(A) receptors compared to their synaptic counterparts.
Publisher: Bentham Science Publishers Ltd.
Date: 31-05-2014
DOI: 10.2174/13816128113199990576
Abstract: A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight. The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5RA21326F
Abstract: Compound 1 is a selective and potent agonist of the G protein-coupled receptor GPR139 (EC 50 = 39 nM).
Publisher: American Chemical Society (ACS)
Date: 30-10-2017
DOI: 10.1021/ACS.JMEDCHEM.7B01351
Abstract: γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K
Publisher: Elsevier BV
Date: 07-2007
DOI: 10.1016/J.GENE.2007.03.008
Abstract: GPRC6A is a novel member of family C of G protein-coupled receptors with so far elusive biological function. GPRC6A has been described in human and mouse as a promiscuous l-alpha-amino acid receptor. We now report the cloning, expression analysis and, functional characterization of the rat orthologue of GPRC6A. Full-length cloning of rat GPRC6A (rGPRC6A) was accomplished using lification of cDNA from taste tissue, and the identity of rGPRC6A confirmed at both the genomic and the protein level by similarity studies. Using selective primers, reverse transcriptase polymerase chain reaction showed that the mRNA is widely but weakly distributed, except for a high expression in the soft palate, the so-called geschmacksstreifen. On the protein level, rGPRC6A was shown to be glycosylated and most likely oligomeric, and using immunochemistry we observed that rGPRC6A is expressed at the plasma membrane of mammalian cell lines. Utilizing co-expression of rGPRC6A and the promiscuous Galpha(q)(G66D) protein in an engineered cell-based inositol phosphate turnover assay, we were able to study the ligand profile of the receptor. We found that l-ornithine is the most potent and efficacious l-amino acid agonist with an EC(50) value of 264 microM, followed by several other aliphatic, neutral, and basic amino acids. Furthermore, the alent cation Mg(2+) was found to be a positive modulator of the l-ornithine response. The presented quantitative pharmacological data underlines the evolutionary conservation of GPRC6A to the rat and signifies the physiological importance and emerging pharmacological potential of GPRC6A.
Publisher: Wiley
Date: 25-06-2020
DOI: 10.1002/GLIA.23872
Abstract: Synaptic transmission is closely linked to brain energy and neurotransmitter metabolism. However, the extent of brain metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the relative metabolic contributions of neurons and astrocytes, are yet unknown. The present study was designed to investigate the functional significance of brain GABA metabolism using isolated mouse cerebral cortical slices and slices of neurosurgically resected neocortical human tissue of the temporal lobe. By using dynamic isotope labeling, with [
Publisher: American Chemical Society (ACS)
Date: 04-10-2013
DOI: 10.1021/JM4011719
Publisher: Springer Science and Business Media LLC
Date: 04-04-2020
DOI: 10.1007/S11064-020-03017-Y
Abstract: Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1 R ,2 S )-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [ 3 H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC 50 values (4.7 and 556 μM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.
Publisher: Springer Science and Business Media LLC
Date: 06-02-2013
DOI: 10.1007/S00726-012-1456-Y
Abstract: GABAA receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure-function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA(A) receptor combinations expressed in Xenopus oocytes using the two-electrode voltage cl technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA(A) receptors, α4/δ-containing GABA(A) receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.
Publisher: Wiley
Date: 18-03-2009
DOI: 10.1111/J.1472-8206.2008.00664.X
Abstract: Gamma-Hydroxybutyric acid (GHB) is a proposed neurotransmitter or neuromodulator with a yet unresolved mechanism of action. GHB binds to both specific high-affinity GHB binding sites and to gamma-aminobutyric acid subtype B (GABA(B)) receptors in the brain. To separate specific GHB effects from GABA(B) receptor effects, it is imperative to develop GHB selective and potent compounds. We generated the compound, 4-(biphen-4-yl)-4-hydroxybutyric acid, which is the 4-hydroxyl analogue of the non-steroidal anti-inflammatory drug (NSAID) fenbufen (referred to as gamma-hydroxyfenbufen). When measured in a rat brain homogenate [(3)H]NCS-382 binding assay, gamma-hydroxyfenbufen inhibited [(3)H]NCS-382 binding with a 10-fold higher affinity than GHB (K(i) 0.44 microM), thus establishing it as a novel lead structure. The active metabolite of fenbufen, 4-biphenylacetic acid inhibited [(3)H]NCS-382 binding with a twofold higher affinity than GHB. Measuring the affinities of structurally related NSAIDs for the [(3)H]NCS-382 site identified diclofenac, a clinically relevant NSAID (Voltaren, Diclon) of the phenylacetic acid (PAA) type, as a GHB ligand (K(i) value of 5.1 microM). Other non-NSAID PAAs also exhibited affinities similar to GHB. Our data raise the interesting possibility that the widely used over-the-counter drug compound, diclofenac, might affect GHB binding at relevant clinical dosages. Furthermore, the identification of PAAs as GHB ligands supplies new information about the structural preferences of the GHB ligand-binding site.
Publisher: American Chemical Society (ACS)
Date: 17-08-2010
DOI: 10.1021/JM1006325
Abstract: Gamma-hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report the design and synthesis of the first (125)I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites.
Publisher: Elsevier BV
Date: 02-2023
Publisher: American Chemical Society (ACS)
Date: 17-01-2013
DOI: 10.1021/JM301656H
Abstract: The isoxazol-3-one tautomer of the bicyclic isoxazole, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol (THAZ), has previously been shown to be a weak GABA(A) and glycine receptor antagonist. In the present study, the potential in this scaffold has been explored through the synthesis and pharmacological characterization of a series of N- and O-substituted THAZ analogues. The analogues N-Bn-THAZ (3d) and O-Bn-THAZ (4d) were found to be potent agonists of the human 5-HT(2A) and 5-HT(2C) receptors. Judging from an elaborate pharmacological profiling at numerous other CNS targets, the 3d analogue appears to be selective for the two receptors. Administration of 3d substantially improved the cognitive performance of mice in a place recognition Y-maze model, an effect fully reversible by coadministration of the selective 5-HT(2C) antagonist SB242084. In conclusion, as novel bioavailable cognitive enhancers that most likely mediate their effects through 5-HT(2A) and/or 5-HT(2C) receptors, the isoxazoles 3d and 4d constitute interesting leads for further medicinal chemistry development.
Publisher: Elsevier BV
Date: 06-2001
Publisher: Wiley
Date: 03-2006
Publisher: Wiley
Date: 04-2006
Publisher: Elsevier BV
Date: 02-2020
DOI: 10.1016/J.EJPS.2019.105203
Abstract: The small-molecule ligand (E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is an analogue of γ-hydroxybutyric acid (GHB) and is widely used for probing the brain-specific GHB high-affinity binding sites. To reach these, brain uptake is imperative, and it is therefore important to understand the molecular mechanisms of NCS-382 transport in order to direct in vivo studies. In this study, we hypothesized that NCS-382 is a substrate for the monocarboxylate transporter subtype 1 (MCT1) which is known to mediate blood-brain barrier (BBB) permeation of GHB. For this purpose, we investigated NCS-382 uptake by MCT subtypes endogenously expressed in tsA201 and MDA-MB-231 cell lines in assays of radioligand-based competition and fluorescence-based intracellular pH measurements. To further verify the results, we measured NCS-382 uptake by means of mass spectrometry in Xenopus laevis oocytes heterologously expressing MCT subtypes. As expected, we found that NCS-382 is a substrate for MCT1 with half-maximal effective concentrations in the low millimolar range. Surprisingly, NCS-382 also showed substrate activity at MCT4 as well as uptake in water-injected oocytes, suggesting a component of passive diffusion. In conclusion, transport of NCS-382 across membranes differs from GHB as it also involves MCT4 and/or passive diffusion. This should be taken into consideration when designing pharmacological studies with this compound and its closely related analogues. The combination of MCT assays used here exemplifies a setup that may be suitable for a reliable characterization of MCT ligands in general.
Publisher: Wiley
Date: 27-02-2014
DOI: 10.1111/BPH.12365
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 18-05-2015
Abstract: γ-Hydroxybutyric acid (GHB) is a recreational drug, a clinically prescribed drug in narcolepsy and alcohol dependence, and an endogenous substance that binds to both high- and low-affinity sites in the brain. For studying the molecular mechanisms and the biologic role of the GHB high-affinity binding sites, ligands with high and specific affinity are essential. The conformationally restricted GHB analog HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid) is one such compound. The objective of this study was to investigate the transport of HOCPCA across the blood-brain barrier in vitro and in vivo and to investigate the hypothesis that HOCPCA, like GHB, is a substrate for the monocarboxylate transporters (MCTs). For in vitro uptake studies, MCT1, -2, and -4 were recombinantly expressed in Xenopus laevis oocytes, and the previously reported radioligand [(3)H]HOCPCA was used as substrate. HOCPCA inhibited the uptake of the endogenous MCT substrate l-[(14)C]lactate, and [(3)H]HOCPCA was shown to act as substrate for MCT1 and 2 (Km values in the low- to mid-millimolar range). Introducing single-point amino acid mutations into positions essential for MCT function supported that HOCPCA binds to the endogenous substrate pocket of MCTs. MCT1-mediated brain entry of HOCPCA (10 mg/kg s.c.) was further confirmed in vivo in mice by coadministration of increasing doses of the MCT inhibitor AR-C141990 [(R)-5-(3-hydroxypyrrolidine-1-carbonyl)-1-isobutyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione], which inhibited brain penetration of HOCPCA in a dose-dependent manner (ID50 = 4.6 mg/kg). Overall, our study provides evidence that MCT1 is an important brain entry site for HOCPCA and qualifies for future in vivo studies with HOCPCA.
Publisher: Wiley
Date: 11-2003
DOI: 10.1111/J.1460-9568.2003.03013.X
Abstract: gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.
Location: France
No related grants have been discovered for Petrine Wellendorph.