GABAA Receptor Expression in the Forebrain of Ataxic Rolling Nagoya Mice.

The human CACNA1A gene encodes the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels and is the locus for several neurological disorders, including episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6) and Familial Hemiplegic Migraine type 1 (FHM1). Several spontaneous mouse Cacna1a mutant strains exist, among them Rolling Nagoya (tgrol), carrying the R1262G point mutation in the mouse Cacna1a gene. tgrol mice display a phenotype of severe gait ataxia and motor dysfunction of the hind limbs. At the functional level, the R1262G mutation results in a positive shift of the activation voltage of the CaV2.1 channel and reduced current density. γ-Aminobutyric acid type A (GABAA) receptor subunit expression depends critically on neuronal calcium influx, and GABAA receptor dysfunction has previously been described for the cerebellum of tgrol and other ataxic Cacna1a mutant mice. Given the expression pattern of CaV2.1, it was hypothesized that calcium dysregulation in tgrol might affect GABAA receptor expression in the forebrain. Herein, functional GABAA receptors in the forebrain of tgrol mice were quantified and pharmacologically dissociated using [3H] radioligand binding. No gross changes to functional GABAA receptors were identified. Future cell type-specific analyses are required to identify possible cortical contributions to the psychomotor phenotype of tgrol mice.

Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4ß2 receptors: unique role of halogen bonding revealed.

The α4ß2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4ß2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4ß2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4ß2 receptors. Crystal structures of five agonists with efficacies at α4ß2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors.

Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K(i) values of around 0.20nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis in an overall yield of approximately 40%, and a library of 9-substituted analogues was prepared by cross-coupling reactions. Compound 8e, 21, 22, and 24 were tested on recombinant rat α(1)ß(3)γ(2), α(2)ß(3)γ(2), α(3)ß(3)γ(2), and α(5)ß(3)γ(2) subtypes, and displayed selectivity for the α(1)ß(3)γ(2) isoform.

Molecular imaging of alpha7 nicotinic acetylcholine receptors: design and evaluation of the potent radioligand [18F]NS10743.

PURPOSE: The outstanding diversity of cellular properties mediated by neuronal and nonneuronal alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) points to the diagnostic potential of quantitative nuclear molecular imaging of alpha7 nAChR in neurology and oncology. It was our goal to radiolabel the alpha7 nAChR agonist 4-[5-(4-fluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-1,4-diaza-bicyclo[3.2.2]nonane (NS10743) and to assess the selectivity of [(18)F]NS10743 binding site occupancy in animal experiments. METHODS: [(18)F]NS10743 was synthesized by nucleophilic substitution of the nitro precursor. In vitro receptor affinity and selectivity were assessed by radioligand competition and autoradiography. The radiotracer properties were evaluated in female CD-1 mice by brain autoradiography and organ distribution. Target specificity was validated after treatment with SSR180711 (10 mg/kg, intraperitoneal), and metabolic stability was investigated using radio-HPLC. RESULTS: The specific activity of [(18)F]NS10743 exceeded 150 GBq/micromol at a radiochemical purity >99%. In vitro, NS10743 and [(18)F]NS10743 showed high affinity and specificity towards alpha7 nAChR. The brain permeation of [(18)F]NS10743 was fast and sufficient with values of 4.83 and 1.60% injected dose per gram and brain to plasma ratios of 3.83 and 2.05 at 5 and 60 min after radiotracer administration. Brain autoradiography and organ distribution showed target-specific accumulation of [(18)F]NS10743 in brain substructures and various alpha7 nAChR-expressing organs. The radiotracer showed a high metabolic stability in vivo with a single polar radiometabolite, which did not cross the blood-brain barrier. CONCLUSION: The good in vitro and in vivo features of [(18)F]NS10743 make this radioligand a promising candidate for quantitative in vivo imaging of alpha7 nAChR expression and encourage further investigations.

Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABA(A) receptors.

A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed on GABA(A) receptor subtypes (alpha(1)beta(3)gamma(2s), alpha(2)beta(3)gamma(2s), alpha(4)beta(3)gamma(2s) and alpha(5)beta(3)gamma(2s)), displaying nanomolar affinities as well as selectivity for alpha1- versus alpha2- and alpha3-containing receptors by a factor of between 14 and 26.

Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity.

Pre-synaptic dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT) terminate synaptic catecholamine transmission through reuptake of released neurotransmitter. Common approaches for studying these transporters involve radiolabeled substrates or inhibitors which, however, have several limitations. In this study we have used a novel neurotransmitter transporter uptake assay kit. The assay employs a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity. In order to validate the assay, a variety of reference and proprietary neurotransmitter transporter ligands from a number of chemical and pharmacological classes were tested. The ability of these compounds to inhibit the selective transporter-mediated uptake demonstrated a similar rank order of potency and IC(50) values close to those obtained in radiolabeled neurotransmitter uptake assays. The described assay enables monitoring of dynamic transport activity of DAT, NET and SERT and is amenable for high-throughput screening and compound characterization.

New ligands with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors. Synthesis, receptor binding, and 3D-QSAR modeling.

A new series of piperazines, diazepanes, diazocanes, diazabicyclononanes, and diazabicyclodecanes with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors were synthesized on the basis of results from a previous computational study. A predictive 3D-QSAR model was developed using the GRID/GOLPE approach (R2 = 0.94, Q2 = 0.83, SDEP = 0.34). The SAR was interpreted in terms of contour maps of the PLS coefficients and in terms of a homology model of the alpha4beta2 subtype of the nicotinic acetylcholine receptors. The results reveal that hydrogen bonding from both hydrogens on the protonated amine and from the pyridine nitrogen to a water molecule as well as van der Waals interactions between the substituent bearing the protonated amine and the receptor is of importance for ligand affinity. The combination of 3D-QSAR and homology modeling proved successful for the interpretation of structure-affinity relationships as well as the validation of the individual modeling approaches.

4-quinolone derivatives: high-affinity ligands at the benzodiazepine site of brain GABA A receptors. synthesis, pharmacology, and pharmacophore modeling.

The 3-ethoxycarbonyl-4-quinolone compound 1 has previously been identified via a database search as an interesting lead compound for ligand binding at the benzodiazepine site of GABA(A) receptors (Kahnberg et al. J. Mol. Graphics Modelling 2004, 23, 253-261). Pharmacophore-guided optimization of this lead compound yielded a number of high-affinity ligands for the benzodiazepine site including compounds 20 and 23-25 displaying sub-nanomolar affinities. A few of the compounds have been tested on the alpha(1)beta(2)gamma(2S) and alpha(3)beta(2)gamma(2S) GABA(A) receptor subtypes, and two of the compounds (5 and 19) display selectivity for alpha(1)- versus alpha(3)-containing receptors by a factor of 22 and 27, respectively. This selectivity for alpha(1)beta(2)gamma(2S) is in the same range as that for the well-known alpha(1) subunit selective compound zolpidem.

The use of a pharmacophore model for identification of novel ligands for the benzodiazepine binding site of the GABAA receptor.

A Catalyst pharmacophore model has been developed for the benzodiazepine site within the GABA(A) receptor complex. The model is based on a pharmacophore model originally proposed by Cook and co-workers (Drug Des. Discovery 1995, 12, 193-248) and further developed by Kahnberg et al. (J. Med. Chem. 2002, 45, 4188-4201). The Catalyst pharmacophore model has been validated by using a series of flavonoids with varying affinities for the benzodiazepine receptor and has then been used as a search query in database searching with the aim of finding novel structures which have the possibility to be modified into novel lead compounds. Five of the hits from the database searching were purchased and their affinities for the benzodiazepine site of the GABA(A) receptor were determined. Two of the compounds displayed K(i) values below 10 microM. The substance showing highest potency in-vitro displayed an affinity of 121 nM making it an interesting compound for optimization. The false positive compounds (K(i) values >10 microM affinities) have been analysed in terms of conformational energy penalties and possibilities for hydrogen bond interactions. The analysis clearly demonstrates the need for post processing of Catalyst hits.

New series of morpholine and 1,4-oxazepane derivatives as dopamine D4 receptor ligands: synthesis and 3D-QSAR model.

Since the identification of the dopamine D(4) receptor subtype and speculations about its possible involvement in schizophrenia, much work has been put into development of selective D(4) ligands. These selective ligands may be effective antipsychotics without extrapyramidal side effects. This work describes the synthesis of a new series of 2,4-disubstituted morpholines and 2,4-disubstituted 1,4-oxazepanes with selectivity for the dopamine D(4) receptor. A 3D-QSAR analysis using the GRID/GOLPE methodology was performed with the purpose to get a better understanding of the relationship between chemical structure and biological activity. Inspection of the coefficient plots allowed us to identify that regions which are important for affinity are situated around the two benzene ring systems, a p-chlorobenzyl group, and the aliphatic amine belonging to the morpholine or 1,4-oxazepane system. In addition, the size of the morpholine or 1,4-oxazepane ring seems to be important for affinity.