Human A3 Adenosine Receptor Research Articles

Association analysis of adenosine A1 receptor gene (ADORA1) polymorphisms with schizophrenia in a Japanese population

The human adenosine A1 receptor gene (ADORA1) localizes to chromosome 1q32 is 76.8 kbp in length and contains six exons. ADORA1 is ubiquitously expressed in the central nervous system and clinical and pharmacological evidence suggest the involvement of adenosine neurotransmission in the pathogenesis of schizophrenia. Therefore, we investigated the contribution of genetic variations of ADORA1 to the pathophysiological mechanisms of Japanese schizophrenia patients. We performed genetic analysis of 29 polymorphic markers in 200 schizophrenic patients and 210 healthy controls from the Kyushu region of Japan. In statistical analysis, we performed the univariate analysis with genotypes and allele frequencies, linkage disequilibrium (LD) analyses, multivariate analysis, haplotype analysis, and sliding window haplotype analysis. In univariate analysis, no statistical difference was shown, after Bonferroni correction. By LD analysis, however, we could not find any LD blocks. In haplotype analysis, a total of 359 haplotypes were estimated. In multivariate analysis, we found three statistically different markers. In sliding window haplotype analysis, there were four statistically different haplotypes. This is the first study describing the involvement of ADORA1 polymorphisms in the pathophysiological mechanisms of schizophrenia in a Japanese population. These results corroborate our previous pharmacological and neurochemical studies in the rat that have suggested an association between ADORA1 neurotransmission and the schizophrenic effects of the N-methyl-D-aspartate receptor antagonist phencyclidine. Thus, ADORA1 polymorphisms may represent good candidate markers for schizophrenia research and ADORA1 may be involved in the pathophysiological mechanisms of schizophrenia in Japanese populations.

The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor

Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A3 (hA3R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([125I]N6-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) – [125I] AB MECA) binding at the hA3R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [125I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA3R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA3R and the basal signalling of this receptor. Since the hA3R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.

Computational study of the molecular mechanisms of caffeine action: Caffeine complexes with adenosine receptors

AbstractTo understand the molecular basis of the principal biological action of the caffeine (CAF), the molecular mechanics calculations of possible complexes between CAF and the fragments of human A1 adenosine receptor were performed. The fragments were selected after considerations of the CAF molecular structure and its possible interactions, as well as after an analysis of the extensive bibliography on the structure, biological role, site‐directed mutagenesis, and the modeling of the adenosine receptors. The minimum energy configurations of these complexes were obtained using two different computer programs with different force fields. The most favorable configurations correspond to the formation of two hydrogen bonds between the CAF molecule and hydrophilic amino acid residues of the fragments of transmembrane domains of the receptor. These configurations are supposed to contribute to CAF blocking of the adenosine receptors. They will be used later for the construction of model CAF complexes with two transmembrane domains simultaneously. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one as a New Scaffold To Develop Potent and Selective Human A3 Adenosine Receptor Antagonists. Synthesis, Pharmacological Evaluation, and Ligand−Receptor Modeling Studies

The paper describes a new class of human (h) A(3) adenosine receptor antagonists, the 2-arylpyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one derivatives (PTP), either 4-oxo (1-6, series A) or 4-amino-substituted (7-20, series B). In both series A and B, substituents able to act as hydrogen bond acceptors (OMe, OH, F, COOEt) were inserted on the 2-phenyl ring. In series B, cycloalkyl and acyl residues were introduced on the 4-amino group. Some of the new derivatives showed high hA(3) AR affinities (K(i) < 50 nM) and selectivities vs both hA(1) and hA(2A) receptors. The selected 4-benzoylamino-2-(4-methoxyphenyl)pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one (18), tested in an in vitro rat model of cerebral ischemia, proved to be effective in preventing the failure of synaptic activity induced by oxygen and glucose deprivation in the hippocampus. Molecular docking of this new class of hA(3) AR antagonists was carried out to depict their hypothetical binding mode to our refined model of hA(3) receptor.

Live Cell Imaging Of The Kinetics Of Ligand Binding At The Human Adenosine A3 Receptor

The aim of this study was to investigate the association and dissociation kinetics of fluorescently labelled adenosine ligands at the human adenosine A3 receptor at a single cell level. CHO cells stably expressing the human adenosine A3 receptor were exposed to 100 nM of fluorescent ligand after which cells were washed with buffer alone or in the presence of an unlabelled adenosine ligand. Confocal fluorescence and phase images were obtained using a Zeiss 510 confocal microscope.The association of ABA-X-BY630, a novel N6-aminoalkyl derivative of adenosine which incorporates the BODIPY [630/650] fluorophore, was monophasic with an association rate constant, kon, of 574700 ± 19000 M−1sec−1, n=7. ABA-X-BY630 dissociation was determined under conditions reflecting that of infinite dilution in the absence and presence of the selective adenosine A3 antagonist, MRS 1220. Under both conditions, ABA-X-BY630 dissociation was monophasic, however the dissociation rate in the absence of antagonist (koff = 0.019 ± 0.001 sec−1, n=4) was significantly slower than that in the presence of 1 μM MRS 1220 (koff = 0.080 ± 0.007 sec−1, n=4).In summary, confocal imaging has been used to directly measure, at single cell level, the binding kinetics of the fluorescent adenosine agonist, ABA-X-BY630. In addition, the perfusion system allows for the rapid removal of ligand and as such the comparison of ABA-X-BY630 dissociation in the absence and presence of antagonist. Under infinite dilution conditions, the dissociate rate of ABA-X-BY630 should be unaffected by the presence of a simple competitive antagonist. Therefore the ability of MRS 1220 to enhance the dissociation rate of ABA-X-BY630 suggests that there may be a negatively cooperative interaction occurring between the two ligands. Similar experiments have also been performed using additional fluorescently labelled adenosine ligands.

A Series of 2,4-Disubstituted Quinolines as a New Class of Allosteric Enhancers of the Adenosine A3 Receptor

The adenosine receptor subfamily consists of the adenosine A(1), A(2A), A(2B), and A(3) receptors, which are localized in a variety of tissues throughout the human body. It is, therefore, a challenge to develop receptor specific ligands with improved tissue selectivity. Allosteric modulators could have these therapeutic advantages over orthosteric ligands. In the present study, a series of 2,4-disubstituted quinolines were synthesized on the basis of the structure of LUF6000 (34). Compound 27 (LUF6096) was able to allosterically enhance agonist binding to a similar extent as 34. In addition, this new compound showed low, if any, orthosteric affinity for any of the adenosine receptors. In a functional assay, compound 27 showed improved activity in comparison to 34, as it increased both the intrinsic efficacy and the potency of the reference agonist Cl-IB-MECA at the human adenosine A(3) receptor.

Flexible modulation of agonist efficacy at the human A3 adenosine receptor by the imidazoquinoline allosteric enhancer LUF6000

BackgroundA series of 1H-imidazo- [4,5-c]quinolin-4-amine derivatives, represented by LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo [4,5-c]quinolin-4-amine), are allosteric modulators of the human A3 adenosine receptor (AR). Here we studied the modulation by LUF6000 of the maximum effect (Emax) of structurally diverse agonists at the A3 AR stably expressed in CHO cells.ResultsIn an assay of [35S]GTPγS binding, the Emax of the A3 AR agonist Cl-IB-MECA at the A3 AR was lower than that of the non-selective AR agonist NECA. LUF6000 exerted an Emax-enhancing effect at a concentration of 0.1 μM or higher, and was shown to increase the Emax of Cl-IB-MECA and other low-efficacy agonists to a larger extent than that of the high-efficacy agonist NECA. Interestingly, LUF6000 converted a nucleoside A3 AR antagonist MRS542, but not a non-nucleoside antagonist MRS1220, into an agonist. LUF6000 alone did not show any effect. Mathematical modeling was performed to explain the differential effects of LUF6000 on agonists with various Emax. A simple explanation for the observation that LUF6000 has a much stronger effect on Cl-IB-MECA than on NECA derived from the mathematical modeling is that NECA has relatively strong intrinsic efficacy, such that the response is already close to the maximum response. Therefore, LUF6000 cannot enhance Emax much further.ConclusionLUF6000 was found to be an allosteric enhancer of Emax of structurally diverse agonists at the A3 AR, being more effective for low-Emax agonists than for high-Emax agonists. LUF6000 was demonstrated to convert an antagonist into an agonist, which represents the first example in G protein-coupled receptors. The observations from the present study are consistent with that predicted by mathematical modeling.

Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

BackgroundAn approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced.ResultsA known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N6-chain elongated functionalized congener ADAC (N6-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (Ki app = 2.4 nM) and functional assays (EC50 = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A1 AR subtype, but there was no selectivity for the A3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A3 receptor but did not bind cells that did not express the receptor.ConclusionThis is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A3 AR dimers.

Synthesis of 2-alkynyl substituted 4′-thioadenosine derivatives and their binding affinities at the adenosine receptors

On the basis of high binding affinity of 2-hexynyl-N(6)-methyladenosine and N(6)-substituted-4'-thioadenosine derivatives at the A3 adenosine receptor (AR), novel 2-alkynyl-substituted-N(6)-methyl-4'-thioadenosine derivatives, combining the characteristics of two classes of nucleosides were designed and synthesized from D-gulonic gamma-lactone via palladium-catalyzed cross coupling reaction as a key step. Among compounds tested, only compound 3b showed moderate binding affinity at the human A3 adenosine receptor without binding affinities at other subtypes.

Synthesis of 3′-Acetamidoadenosine Derivatives as Potential A3 Adenosine Receptor Agonists

On the basis of high binding affinity of 3′-aminoadenosine derivatives 2b at the human A3 adenosine receptor (AR), 3′-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A3AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A3AR.

Quinolinedione nucleus as a novel scaffold for A1 and A2A adenosine receptor antagonists

In the last few years, much effort has been directed towards the synthesis of selective adenosine receptor (AR) antagonists since they are attractive tools for pharmacological intervention in many pathophysiological conditions. During our studies aimed at obtaining new nonclassical adenosine antagonists devoid of phosphodiesterase (PDE) inhibition, a series of 2-pyridones and 2,5-quinolinediones (3a–f, 5a–f, 6a,c–f) has been synthesized as potential AR ligands. Binding affinities of the new compounds were determined for bovine and human adenosine A1, A2A, and A3 receptors. Compound 5f showed good affinity (K i = 7.8 μM) towards human A1AR but no selectivity (K i = 7.0 μM) towards human A2AAR, whereas compound 6f showed more affinity towards human A2A (K i = 16 μM) than A1 receptor (percentage inhibition at 10 μM concentration = 11). In the 1–100 μM range, the new compounds did not inhibit cardiac PDE3 activity at all. Molecular modeling studies carried out on 5f and 6f support the pharmacological results and suggest 6f as a potential lead compound selective towards A2AAR.

Characterisation of the adenosine A1 receptor in Saccharomyces cerevisiae

Different ligands for G protein‐coupled receptors can induce unique receptor conformations, leading to differing profiles of G protein coupling. Given that S. cerevisiae expression systems are useful tools for studying the coupling of GPCRs to individual G protein pathways, experiments were undertaken to investigate whether different pharmacological profiles can be detected in this system for ligands of the adenosine A1 receptor (A1R), and to what extent the findings correlated to a mammalian (CHO) expression system. The human A1R was introduced into individual strains of S. cerevisiae, each expressing different chimeras from yeast and mammalian Gα subunits, followed by pharmacological characterisation. We found that, in the yeast, the A1R only couples to Gαi/o family proteins when stimulated with the full agonist, R‐PIA. The pA2 values generated from analysis of the interaction between the A1R antagonist, DPCPX, and R‐PIA correlated with that observed in mammalian cell assays. A novel partial agonist at the A1R, VCP‐189, was also tested. VCP‐189 appeared to selectively activate Gαi subtypes, but no response was seen with Gαo or Gαz strains. In agreement with the S. cerevisiae assay, the response to R‐PIA and VCP‐189 in the mammalian assays is pertussis toxin sensitive. Further studies investigating Gαi/o subtype selectivity in mammalian cells are currently underway.

Linear and Nonlinear 3D-QSAR Approaches in Tandem with Ligand-Based Homology Modeling as a Computational Strategy To Depict the Pyrazolo-Triazolo-Pyrimidine Antagonists Binding Site of the Human Adenosine A2A Receptor

The integration of ligand- and structure-based strategies might sensitively increase the success of drug discovery process. We have recently described the application of Molecular Electrostatic Potential autocorrelated vectors (autoMEPs) in generating both linear (Partial Least-Square, PLS) and nonlinear (Response Surface Analysis, RSA) 3D-QSAR models to quantitatively predict the binding affinity of human adenosine A3 receptor antagonists. Moreover, we have also reported a novel GPCR modeling approach, called Ligand-Based Homology Modeling (LBHM), as a tool to simulate the conformational changes of the receptor induced by ligand binding. In the present study, the application of both linear and nonlinear 3D-QSAR methods and LBHM computational techniques has been used to depict the hypothetical antagonist binding site of the human adenosine A2A receptor. In particular, a collection of 127 known human A2A antagonists has been utilized to derive two 3D-QSAR models (autoMEPs/PLS&RSA). In parallel, using a rhodopsin-driven homology modeling approach, we have built a model of the human adenosine A2A receptor. Finally, 3D-QSAR and LBHM strategies have been utilized to predict the binding affinity of five new human A2A pyrazolo-triazolo-pyrimidine antagonists finding a good agreement between the theoretical and the experimental predictions.

Structure–activity relationships of 2-chloro-N6-substituted-4′-thioadenosine-5′-N,N-dialkyluronamides as human A3 adenosine receptor antagonists

On the basis of potent and selective A(3) adenosine receptor (AR) antagonist, 2-chloro-N(6)-(3-iodobenzyl)-4'-thioadenosine-5'-N,N-dimethyluronamide, structure-activity relationships were studied for a series of 5'-N,N-dialkyluronamide derivatives, synthesized from D-gulonic gamma-lactone. From this study, it was revealed that removal of the hydrogen bond-donating ability of the 5'-uronamide was essential for the pure A(3)AR antagonism. 5'-N,N-Dimethyluronamide derivatives exhibited higher binding affinity than larger 5'-N,N-dialkyl or 5'-N,N-cycloalkylamide derivatives, indicating that steric factors are crucial in binding to the human A(3)AR. A N(6)-(3-bromobenzyl) derivative 6c (K(i)=9.32 nM) exhibited the highest binding affinity at the human A(3)AR with very low binding affinities to other AR subtypes.

Synthesis and Biological Activities of 4-Phenyl-5-pyridyl-1,3-thiazole Derivatives as Selective Adenosine A3 Antagonists

To investigate the potency of an adenosine A3 receptor (A3AR) antagonist as an anti-asthmatic drug, a novel series of 4-phenyl-5-pyridyl-1,3-thiazole derivatives was synthesized and evaluated in human adenosine A1, A2A and A3 receptor and rat adenosine A3 receptor binding assays. From investigation of the SAR study, compound 7af was identified as a highly potent human and rat A3AR antagonist. This compound inhibited IB-MECA-induced plasma protein extravasation in the skin of rats and showed good oral absorption. Also, compound 7af significantly inhibited antigen-induced hyper-responsiveness to acetylcholine in actively sensitized Brown Norway rats. These results show that 4-phenyl-5-pyridyl-1,3-thiazole derivatives are potential candidates to enable the evaluation of A3AR antagonists. Further evaluation of this class of compounds may afford a novel anti-inflammatory agent such as an anti-asthmatic drug.

Scouting Human A3 Adenosine Receptor Antagonist Binding Mode Using a Molecular Simplification Approach: From Triazoloquinoxaline to a Pyrimidine Skeleton as a Key Study

The concept of molecular simplification as a drug design strategy to shorten synthetic routes, while keeping or enhancing the biological activity of the lead drug, has been applied to design new classes of human A3 adenosine receptor (AR) antagonists. Over the past decade, we have focused a part of our research on the study of AR antagonists belonging to strictly correlated classes of tricyclic compounds. One of these classes is represented by the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, either 4-amino or 4-oxo-substituted, which were intensively investigated by evaluating the effect of different substituents on the 2-phenyl ring and on the 4-amino group. Using an in silico molecular simplification approach, a new series of easily synthesizable 2-amino/2-oxoquinazoline-4-carboxamido derivatives have been discovered, presenting high affinity and selectivity against human A3 AR.

Design, Synthesis, and Anti-Tumor Activity of 4′-Thionucleosides as Potent and Selective Agonists at the Human A3 Adenosine Receptor

On the basis of potent and selective binding affinity of Cl-IB-MECA to the human A3 adenosine receptor, its 4′-thioadenosine derivatives were efficiently synthesized starting from D-gulonic γ -lactone. Among compounds tested, 2-chloro-N 6-(3-iodobenzyl)- and 2-chloro-N 6-methyl-4′ -thioadenosine-5′ -methyluronamides (7a and 7b) exhibited nanomolar range of binding affinity (K i = 0.38 nM and 0.28 nM, respectively) at the human A3AR. These compounds showed anti-growth effects on HL-60 leukemia cell, which resulted from the inhibition of Wnt signaling pathway.

Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells.

G protein-coupled receptors are known to be organized within different membrane compartments or microdomains of individual cells. Here, we have used a fluorescent A3 adenosine receptor (A3-AR) agonist, ABEA-X-BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist-occupied A3-AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A3-AR, the fluorescent A3-AR agonist was able to inhibit forskolin-stimulated [3H]cAMP production (pEC50=8.57), and this was antagonized by the A3-selective antagonist MRS1220 (pK(B)=9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC50=7.34). Ligand binding to the A3-AR on the membranes of single cells and subsequent increases in single cell [Ca2+]i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small-membrane microdomains (approximately 0.2 microm2) revealed two agonist-occupied A3-AR components with differing diffusion characteristics (diffusion coefficients=2.65x10(-8) and 1.19x10(-9) cm2/s, respectively). The binding of ligand to these two components was reduced from 5.1 and 14.9 to 2.6 and 3.3 receptors/microm2, respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist-occupied A3-receptor complexes, showing different motilities within the membrane of single living cells.

5-Amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one: A Versatile Scaffold To Obtain Potent and Selective A3 Adenosine Receptor Antagonists

Binding assays on human A1, A2A, and A3 adenosine receptors (ARs) and functional studies on A2B ARs revealed that various 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones VIII, previously reported as ligands at the central benzodiazepine receptor (BzR), possess nanomolar affinity at the A3 AR. Replacement of the amide of VIII with an amidine moiety gave the 5-amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-ones IX, which maintain a nanomolar potency at the A3 AR with selectivity over the BzR. Insertion of a p-methoxybenzoyl at the 5-amino moiety enhanced A3 AR affinity and selectivity over the A1, A2A, and A2B ARs. The best result of our lead optimization efforts is 9-chloro-5-(4-methoxybenzoyl)amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one (23), which displayed a Ki of 1.6 nM at the A3 AR and no significant affinity at the other ARs or the BzR. Docking simulations on selected ligands into a model of the A3 AR allowed us to rationalize the structure-activity relationships of phenyltriazolobenzotriazindiones VIII and aminophenyltriazolobenzotriazinones IX at the molecular level.

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile.

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A1, A2A and A3 adenosine receptors and in adenylyl cyclase experiments for their potency at the human A2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N6 position of 9-propyladenine significantly increased binding affinity at the human A1 and A3 adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N6 position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.