Modulation of Antagonist Binding to Serotonin1A Receptors from Bovine Hippocampus by Metal Ions

1. The serotonin_1A(5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Although specific 5-HT_1Aagonists have been discovered more than a decade back, the development of selective 5-HT_1Aantagonists has been achieved only recently.2. We have examined the modulation of the specific antagonist [³H]p-MPPF binding to 5-HT_1Areceptors from bovine hippocampal membranes by monovalent and divalent metal ions. Our results show that the antagonist binding to 5-HT_1Areceptors is inhibited by both monovalent and divalent cations in a concentration-dependent manner. This is accompanied by a concomitant reduction in binding affinity.3. Our results also show that the specific antagonist p-MPPF binds to all available receptors in the bovine hippocampal membrane irrespective of their state of G-protein coupling and other serotonergic ligands such as 5-HT and OH-DPAT effectively compete with the specific antagonist [³H]p-MPPF.4. These results are relevant to ongoing analyses of the overall modulation of ligand binding in G-protein-coupled seven transmembrane domain receptors.     

Ligand Binding Characteristics of the Human Serotonin<Subscript>1<Emphasis Type="Italic">A</Emphasis></Subscript> Receptor Heterologously Expressed in CHO Cells

The serotonin_1A (5-HT_1A) receptors are important members of the superfamily of seven transmembrane domain G-protein coupled receptors. They appear to be involved in various behavioral, cognitive and developmental functions. Mammalian cells in culture heterologously expressing membrane receptors represent convenient systems to address problems in receptor biology. We report here the pharmacological characterization of one of the first isolated clones of CHO cells stably expressing the human 5-HT_1A receptor using the selective agonist 8-OH-DPAT and antagonist p-MPPF. In addition, we demonstrate that agonist and antagonist binding to the receptor exhibit differential sensitivity to the non-hydrolyzable GTP analogue, GTP--S, as was observed earlier with the native receptor from bovine hippocampus. These results show that the human 5-HT_1A receptor expressed in CHO cells displays characteristic features found in the native receptor isolated from bovine hippocampus and promises to be a potentially useful system for future studies of the receptor.These authors have contributed equally to the work     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1A receptor

The serotonin_1A (5-HT_1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT_1A receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT_1A receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT_1A receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT_1A receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT_1A receptor function.     

The Human Serotonin 1A Receptor Expressed in Neuronal Cells: Toward a Native Environment for Neuronal Receptors

1. The serotonin_1A (5-HT_1A) receptor is an important representative of G-protein coupled family of receptors. It is the most extensively studied among the serotonin receptors, and appears to be involved in various behavioral and cognitive functions.2. We report here the pharmacological and functional characterization of the human serotonin_1A receptor stably expressed in HN2 cell line, which is a hybrid cell line between hippocampal cells and mouse neuroblastoma.3. Our results show that serotonin_1A receptors in HN2-5-HT_1AR cells display ligand-binding properties that closely mimic binding properties observed with native receptors. We further demonstrate that the differential discrimination of G-protein coupling by the specific agonist and antagonist, a hallmark of the native receptor, is maintained for the receptor in HN2-5-HT_1AR cells. Importantly, the serotonin_1A receptor in HN2-5-HT_1AR cells shows efficient downstream signalling by reducing cellular cyclic AMP levels.4. We conclude that serotonin_1A receptors expressed in HN2-5-HT_1AR cells represent a useful model system to study serotonin_1A receptor biology, and is a potential system for solubilization and purification of the receptor in native-like membrane environment.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

BMY 7378: Partial agonist at spinal cord 5-HT1A receptors

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.     

Ligand Binding and G-protein Coupling of the Serotonin1A Receptor in Cholesterol-enriched Hippocampal Membranes

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Since a large portion of such transmembrane receptors remains in contact with the membrane lipid environment, lipid–protein interactions assume importance in the structure-function analysis of such receptors. We have earlier reported the requirement of cholesterol for serotonin_1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl- β-cyclodextrin. In this paper, we monitored the serotonin_1A receptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-β-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin_1A receptor do not exhibit significant difference in native and cholesterol-enriched hippocampal membranes indicating that further enrichment of cholesterol has little functional consequence on the serotonin_1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin_1A receptor function.     

Role of Disulfides and Sulfhydryl Groups in Agonist and Antagonist Binding in Serotonin1A Receptors from Bovine Hippocampus

SUMMARY 1. The serotonin_1A (5-HT_1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT_1A receptors from bovine hippocampus.2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT_1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity.3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites.4. In addition, ligand-binding studies in presence of GTP--S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT_1A receptor and the G-protein.5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT_1A receptors.     

8-[3H]Hydroxy-2-(di-n-propylamino) tetralin binding sites in goldfish retina

The binding sites of 8-[³H]hydroxy-2-(di-n-propylamino)tetralin ([³H]DPAT) were characterized in the retina of goldfish in order to evaluate the selectivity of the ligand for serotonin_1A (5HT_1A) receptors. Specificity of the binding was performed in the presence of serotonergic and dopaminergic agonists and antagonists. Buspirone, spriroxatrine and 5-methoxy-N,N-dimethyltryptamine were potent inhibitors, followed by propranolol, citalopram, imipramine and desipramine. Serotonin was not a potent inhibitor, and its interaction with the binding sites of [³H]DPAT was complex. Nomifensine displayed an important inhibition, however, other dopamine uptake blockers, such as bupropion and GBR-12909, were less potent. Haloperidol was also a good inhibitor, but the D₁ receptor agonist, SKF-38393, the D₂ receptor antagonist, sulpiride, and dopamine did not inhibit the binding. GppNHp inhibited the binding in the micromolar range. The analysis of saturation experiments by isotopic dilution, using buspirone to determine nonspecific binding, revealed two sites. The number of binding sites defined by buspirone were higher than the ones defined by nomifesine. The specific binding, using buspirone for definition, was reduced by the intraocular injection of 6-hydroxydopamine. This investigation demonstrates that [³H]DPAT labels 5HT_1A receptors in goldfish retina, but also interacts with a non-5HT receptor site. These receptors seem to be localized in dopaminergic neurons.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Membrane Organization and Function of the Serotonin<Subscript>1A</Subscript> Receptor

(1) The serotonin_1A receptor is a G-protein coupled receptor involved in several cognitive, behavioral, and developmental functions. It binds the neurotransmitter serotonin and signals across the membrane through its interactions with heterotrimeric G-proteins. (2) Lipid–protein interactions in membranes play an important role in the assembly, stability, and function of membrane proteins. The role of membrane environment in serotonin_1A receptor function is beginning to be addressed by exploring the consequences of lipid manipulations on the ligand binding and G-protein coupling of serotonin_1A receptors, the ability to functionally solubilize the serotonin_1A receptor, and the factors influencing the membrane organization of the serotonin_1A receptor. (3) Recent developments involving the application of detergent-based and detergent-free approaches to understand the membrane organization of the serotonin_1A receptor under conditions of ligand activation and modulation of membrane lipid content, with an emphasis on membrane cholesterol, are described.     

[11C]WAY 100635: A radioligand for imaging 5-HT1A receptors with positron emission tomography

The potent and selective 5-HT_1A antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide) was radiolabeled with ¹¹C in high specific activity, and the in vivo properties of this radioligand were assessed in the brains of rats and monkeys. Following i.v. tail vein injection in rats, [¹¹C]WAY 100635 rapidly penetrated into brain tissue and was retained over a 30–90 min time period in a manner consistent with the known distribution of 5-HT_1A receptors. Pretreatment of rats with the selective 5-HT_1A agonist (±)-8-OH-DPAT effectively blocked the retention of radioactivity in brain regions known to contain high densities of 5-HT_1A receptors. The hippocampus-to-cerebellum radioactivity concentration ratio reached a maximum of 16:1 at 60 min post injection. Following i.v. injection of [¹¹C]WAY 100635 in rhesus monkeys, the concentrations of radioactivity in brain regions were consistent with the reported distribution of 5-HT_1A receptors in primates, and the frontal cortex-to-cerebellum ratio reached 5.5:1 at 80 min post injection. Pretreatment of the monkeys with (±)-8-OH-DPAT reduced this ratio to 1.4:1, and injection of (±)-8-OH-DPAT 20 min after the injection of [¹¹C]WAY 100635 significantly displaced frontal cortex binding. The in vivo properties of [¹¹C]WAY 100635 in rats and monkeys strongly support the future utility of this radioligand for imaging 5-HT_1A receptors using positron emission tomography (PET).     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of α_2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [³⁵S]-guanosine 5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS) binding assay. α_2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [³⁵S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat α₂ adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the α_2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948) to inhibit agonist’s effect, allowed us to filtrate out α_2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

Heterogeneity of Cortical and Hippocampal 5-HT1A Receptors: A Reappraisal of Homogenate Binding with 8-[3H]Hydroxydipropylaminotetralin

Abstract: The selective serotonin (5-HT) agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5-HT_1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [³H]8- OH-DPAT (free ligand from 200 pM to 160 nM) revealed high-affinity 5-HT_1A receptors (K_H= 0.7–0.8 nM) and lowaffinity (K_L= 22–36 nM) binding sites. The kinetics of [³H]8-OH-DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high- and low-affinity binding sites. When the high-affinity sites were labeled with a 1 nM concentration of [³H]8- OH-DPAT, the competition curves of agonist and antagonist drugs were best fit to a two-site model, indicating the presence of two different 5-HT_1A binding sites or, alternatively, two affinity states, tentatively designated as 5-HT_1AHIGH and 5-HT_1A^LOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5-HT_1A sites are modulated by 5′-guanylylimidodiphosphate, inhibition experiments with 5-HT were performed in the presence or in the absence of 100 μM 5′-guanylylimidodiphosphate. The binding of 1 nM [³H]8-OH-DPAT to the 5-HT_1A^HIGH site was dramatically (80%) reduced by 5′-guanylylimidodiphosphate; in contrast, the low-affinity site, or 5-HT_1A^LOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high-affinity 5-HT_1A^HIGH site corresponds to the classic 5-HT_1A receptor, whereas the novel 5-HT_1A^LOW binding site, labeled by 1 nM [³H]8-OH-DPAT and having a micromolar affinity for 5-HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5-HT_1A sites and the 5-HT innervation, rats were treated with p-chlorophenylalanine or with the neurotoxin p-chloroamphetamine. The inhibition of 5-HT synthesis by p-chlorophenylalanine did not alter either of the two 5-HT_1A sites, but deafferentation by p-chloroamphetamine caused a loss of the low-affinity [³H]8-OH- DPAT binding sites, indicating-that these novel binding sites may be located presynaptically on 5-HT fibers and/or nerve terminals.     

Neuropharmacological profile of a high-affinity ligand of 5-HT1A receptors, 4-phenyl-1-[4-(2-naphtalimido)butyl]-piperazine

The neuropharmacological profile of 4-phenyl-1-[4-(2-naphtalimido)butyl]-piperazine (PNBP), a compound that possesses a high affinity for the serotonin receptors of 1A-type (5-HT_1A) and lacks an anxiolytic action, has been studied. Intracerebral administration of PNBP to rats through implanted cannulae into the hippocampal region resulted in no substantial behavioral changes during “open field” and “conflict situation” tests, as compared with those of control animals. At the same time, the behavioral effects of intraperitoneal administration of 10 mg/kg buspirone were completely abolished if buspirone had been jointly administered with 10 mg/kg PNBP. Moreover, combined application of 0.3 mg/kg PNBP and 0.3 mg/kg 8-OH-DPAT, an agonist of 5-HT_1A receptors, almost completely abolished the components of “serotonin syndrome” (prone position and stamping of the forepaws) in animals under study. These findings allowed us to conclude that PNBP has the properties of a competitive antagonist of buspirone and 8-OH-DPAT.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.     

Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis. This article has previously been published in issue 4/4, under doi:.     

Arylpiperazinylalkyl derivatives of 8-amino-1,3-dimethylpurine-2,6-dione as novel multitarget 5-HT/D receptor agents with potential antipsychotic activity

A series of new 7-arylpiperazinylalkyl-1,3-dimethyl-purine-2,6-dione derivatives with diversified 8-amino substituent in 8 position was synthesized and their 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ receptor affinities were determined. The binding study allowed identifying some potent 5-HT_1A/5-HT_2A/5-HT₇/D₂ ligands. The most interesting because of their multireceptor profile were 8-piperidine (30–35) and 8-dipropylamine (45–47) analogs with four and five carbon aliphatic linkers. The selected compounds 24, 31, 34, 39, 41, 43, 45, and 46 in the functional in vitro evaluation for all targeted receptors showed significant partial D₂ agonist, partial 5-HT_1A agonist, and 5-HT_2A antagonist properties. The advantageous in vitro affinity of compound 34 for 5-HT_1A and D₂ receptors has been explained by means of molecular modeling, taking into consideration its partial agonist activity towards the latter one. In behavioral studies, compounds 32 and 34 revealed antipsychotic-like properties, significantly decreasing d-amphetamine-induced hyperactivity in mice.