Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites.

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.

Design, synthesis, and in vitro pharmacology of new radiolabeled gamma-hydroxybutyric acid analogues including photolabile analogues with irreversible binding to the high-affinity gamma-hydroxybutyric acid binding sites.

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.

Phenylacetic acids and the structurally related non-steroidal anti-inflammatory drug diclofenac bind to specific gamma-hydroxybutyric acid sites in rat brain.

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.

Novel high-affinity and selective biaromatic 4-substituted gamma-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies.

Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4'-phenethylphenyl, 4'-styrylphenyl, and 4'-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding assay and in GABA(A) and GABA(B) receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed Ki values below 100 nM. The affinity of the 4-[4'-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (Ki = 22 nM), which has higher affinity than previously reported GHB ligands.

Structure-activity relationships of selective GABA uptake inhibitors.

For more than four decades there has been a search for selective inhibitors of GABA transporters. This has led to potent and selective inhibitors of the cloned GABA transporter subtype GAT1, which is responsible for a majority of neuronal GABA transport. The only clinically approved compound with this mechanism of action is Tiagabine. Other GABA transporter subtypes have not been targeted with comparable selectivity and potency. We here review a comprehensive series of competitive inhibitors that provide information about the GABA recognition site and summarise the structure-activity relations in a ligand-based pharmacophore model that suggests how future compounds could be designed. Finally, some of the recent results on subtype-characterised competitive inhibitors and recent lipophilic aromatic GABA uptake inhibitors are reviewed.

Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain.

Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.