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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 11 C-labeling and subsequent evaluation of [ 11 C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [ 3 H]HOCPCA. In vivo evaluation of [ 11 C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [ 11 C]HOCPCA.

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 hippocampus 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 hippocampal 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.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

GHB (γ‐hydroxybutyrate) is not only an endogenously present small molecule but also a clinically prescribed drug for the symptomatic treatment of narcolepsy. However, GHB's mechanism of action remains to be uncovered. Within the CNS, GHB targets both GABAB receptors and a pharmacologically distinct population of high‐affinity binding sites with unknown molecular identity. HOCPCA (3‐hydroxycyclopent‐1‐enecarboxylic acid) is a structural analog of GHB selectively targeting GHB high‐affinity binding sites. Here, we discuss the usefulness of 3H‐ and 11C‐labeled HOCPCA as radioligands for selectively probing GHB high‐affinity binding sites and their application in drug discovery. As such, [3H]HOCPCA's exceptional affinity and selectivity makes it an indispensable tool in drug discovery, and its utility has been demonstrated in, for example, homogenate binding studies, in vitro as well as ex vivo autoradiography. Moreover, the successful synthesis of [11C]HOCPCA is a starting point for further ligand development for future in vivo investigations of GHB high‐affinity binding sites. [ABSTRACT FROM AUTHOR]

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 (1R,2S)-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 [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 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. [ABSTRACT FROM AUTHOR]

γ-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 125I-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. [ABSTRACT FROM AUTHOR]

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 Ki) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABA A receptors as possible high-affinity GHB targets. GABA A 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 GABA A receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [ 3 H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABA A receptors, and the naturally occurring flavonoid catechin. These compounds increased [ 3 H]NCS-382 binding to 185–272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [ 3 H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [ 3 H]GHB and the GHB high-affinity site-specific radioligands [ 125 I]BnOPh-GHB and [ 3 H]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 individual 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 GABA A receptors. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

3-Hydroxycyclopent-1-enecarboxylicacid (HOCPCA, 1) is a potent ligand for the high-affinityGHB binding sites in theCNS. An improved synthesis of 1together with a veryefficient synthesis of [3H]-1is described.The radiosynthesis employs in situ generated lithium trimethoxyborotritide.Screening of 1against different CNS targets establishesa high selectivity, and we demonstrate in vivo brain penetration.In vitro characterization of [3H]-1bindingshows high specificity to the high-affinity GHB binding sites. [ABSTRACT FROM AUTHOR]