Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

Angiogenesis is critical to wound repair due to its role in providing oxygen and nutrients that are required to support the growth and function of reparative cells in damaged tissues. Adenosine receptors are claimed to be of paramount importance in driving wound angiogenesis by inducing VEGF. However, the underlying mechanisms for the regulation of adenosine receptors in VEGF as well as eNOS remain poorly understood. In the present study, we found that adenosine and the non-selective adenosine receptor agonists (NECA) induced tube formation in HMEC-1 in a dose-dependent manner. Adenosine or NECA (10 µmol/L) significantly augmented the number and length of the segments in comparison with the control. Simultaneously, VEGF and eNOS were significantly upregulated following the administration of 10 µmol/L NECA, while they were suppressed after A_2B AR genetic silencing and pharmacological inhibition by MRS1754. In addition, VEGF expression and eNOS bioavailability elimination significantly reduced the formation of capillary-like structures. Furthermore, the activation of A_2B AR by NECA significantly increased the intracellular cAMP levels and concomitant CREB phosphorylation, eventually leading to the production of VEGF in HMEC-1. However, the activated PKA-CREB pathway seemed to be invalidated in the induction of eNOS. Moreover, we found that the elicited PI3K/AKT signaling in response to the induction of NECA assisted in regulating eNOS but failed to impact on VEGF generation. In conclusion, the A_2B AR activation-driven angiogenesis via cAMP-PKA-CREB mediated VEGF production and PI3K/AKT-dependent upregulation of eNOS in HMEC-1.     

Discovery of benzothiazole-based adenosine A2B receptor antagonists with improved A2A selectivity

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A_2B antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A_2A and A₁ receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A_2A and A₁ receptors.     

Synthesis and Biological Activity of Trisubstituted Adenines as A 2A Adenosine Receptor Antagonists

The discovery of new drugs for the treatment of neurodegenerative disorders, such as Parkinson's disease, has become an attractive field of research. Due to the regulation of D ₂ receptor activity by A _{2 A} adenosine receptor, potent and selective ligands of A _{2 A} subtype could be useful tools to study neurodegenerative disorders. A series of 2,8-disubstituted-9-ethyladenine derivatives was synthesized and tested in binding affinity assay at human adenosine receptors. New compounds showed good affinity and selectivity at A _{2 A} receptor versus the other subtypes. The introduction of a bromine atom in 8-position increased the affinity of these compounds, leading to ligands with K _i in the nanomolar range.     

氨茶碱与柠檬酸盐协同作用促进环磷酸腺苷发酵生产

目的:探究通过抑制磷酸二酯酶活性促进cAMP发酵合成的工艺方法.方法:在7 L发酵罐上进行添加氨茶碱的发酵实验,通过对发酵主要参数、关键酶活性、能量代谢水平等进行分析,针对性提出了氨茶碱与柠檬酸盐协同作用促进cAMP合成的发酵工艺.结果:与对照相比,添加5 mg/L氨茶碱批次的cAMP产量提高25.9％,副产物腺苷浓度...     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

Design,synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists

A series of novel dual A_2A/A_2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A_2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A_2B AR (IC₅₀ 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A_2A/A_2B AR antagonists for cancer immunotherapy.     

Lack of adenosine A3 receptors causes defects in mouse peripheral blood parameters

The role of the adenosine A₃ receptor in hematopoiesis was studied using adenosine A₃ receptor knockout (A₃AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A₃AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A₃AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A₃AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A₃ receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A₃AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A₃AR KO mice and their WT counterparts.     

α-DNA. Synthesis,Characterization and Base-Pairing Properties of Unnatural α-Oligodeoxyribonucleotides

Abstract

The novel hexadeoxyribonucleotides α-d(CpCpTpTpCpC) and α-d(CpApTpGpCpG), in which each glycosidic linkage exhibit the anomeric α-configuration, were synthesized by the phosphotriester method. ¹H-NMR and thermal denaturation studies provided evidence for these a-oligonucleotides to exhibit a secondary structure similar to that of the natural nucleic acids.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Adenosine A2A receptor deficiency prevents p38MAPK activation and apoptosis of mouse hippocampal cells in the chronic hypoxic-hypercapnia model

ABSTRACT

This study aims to study the effects of adenosine A_2A receptor (A_2AR) on hippocampal cell apoptosis and the putative mechanisms in a mouse model of chronic hypoxic-hypercapnia. Wild-type (WT) or A_2AR knockout (A_2AR KO) mice were randomly divided into normal control (NC) groups and chronic hypoxic-hypercapnia (4HH) groups. Compared with their corresponding NC groups (WT-NC and KO-NC), the apoptosis index (AI), caspase-3 activity, Bax mRNA and P-p38 protein expression in the hippocampus of 4HH groups (WT-4HH and KO-4HH) were significantly increased, while Bcl2 mRNA expression was significantly decreased (P < 0.05). Moreover, A_2AR deficiency significantly rescued the effect of chronic hypoxic-hypercapnia on apoptosis when compared with the WT-4HH group (P < 0.05). A_2AR deficiency inhibits hippocampal cell apoptosis in mice exposed to chronic hypoxic-hypercapnia, which might be associated with dampened p38 MAPK activation and Bax mRNA expression, and augmented Bcl-2 mRNA expression.     

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian G_α subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A_2B receptor (hA_2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA_2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: G_αi, G_αs, G_αq, and G_α12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the G_α subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103^3.50 and I107^3.54 are vital too in G protein-coupling and the activation of the hA_2BR, whereas L213^IL3 is more important in G protein inactivation. Substitution of S235^6.36 to alanine provided the most divergent G protein-coupling profile. Finally, L236^6.37 substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.     

Adenosine A2A receptors assemble into higher-order oligomers at the plasma membrane

Oligomerization of G protein-coupled receptors (GPCRs) is known to play important roles in regulating receptor pharmacology and function. Whereas many bivalent GPCR interactions have been described, the stoichiometry and localization of GPCR oligomers are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to study adenosine A_2A receptor (A_2AR) oligomerization. The data suggest specificity of the A_2AR/A_2AR interaction monitored by BiFC and proper sub-cellular localization of tagged receptors. Moreover, using a novel approach combining fluorescence resonance energy transfer and BiFC, we found that at least three A_2A receptors assemble into higher-order oligomers at the plasma membrane in Cath.A differentiated neuronal cells.

Structured summary

MINT-6797156, MINT-6797142: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by bimolecular fluorescence complementation (MI:0809)

MINT-6797129: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by fluorescent resonance energy transfer (MI:0055)

     

Investigation of the conformational dynamics of the apo A2A adenosine receptor

The activation/deactivation processes for G-protein coupled receptors (GPCRs) have been computationally studied for several different classes, including rhodopsin, the β2 adrenergic receptor, and the M2 muscarinic receptor. Despite determined cocrystal structures of the adenosine A_2A receptor (A_2AAR) in complex with antagonists, agonists and an antibody, the deactivation process of this GPCR is not completely understood. In this study, we investigate the convergence of two apo simulations, one starting with an agonist-bound conformation (PDB: 3QAK)¹⁴ and the other starting with an antagonist-bound conformation (PDB: 3EML)¹¹. Despite the two simulations not completely converging, we were able to identify distinct intermediate steps of the deactivation process characterized by the movement of Y288^7.53 in the NPxxY motif. We find that Y288^7.53 contributes to the process by forming hydrogen bonds to residues in transmembrane helices 2 and 7 and losing these interactions upon full deactivation. Y197^5.58 also plays a role in the process by forming a hydrogen bond only once the side chain moves from the lipid interface to the middle of the helical bundle.     

Synthesis and pharmacological evaluation of novel 1- and 8-substituted-3-furfuryl xanthines as adenosine receptor antagonists

The synthesis of an important set of 3-furfurylxanthine derivatives is described. Binding affinities were determined for rat A₁ and human A_2A, A_2B and A₃ receptors. Several of the 3-furfuryl-7-methylxanthine derivatives showed moderate-to-high affinity at human A_2B receptors, the most active compound (10d) having a K_i of 7.4 nM for hA_2B receptors, with selectivities over rA₁ and hA_2A receptors up to 14-fold and 11-fold, respectively. Affinities for hA₃ receptors were very low for all members of the set.     

Potent and selective adenosine A(2A) receptor antagonists: [1,2,4]-triazolo[4,3-c]pyrimidin-3-ones

Antagonism of the adenosine A_2A receptor affords a possible treatment of Parkinson’s disease. In the course of investigating pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine A_2A antagonists, we prepared [1,2,4]-triazolo[4,3-c]pyrimidin-3-ones with potent and selective (vs A₁) A_2A antagonist activity. Structure-activity relationships are described for this series.     

Stimulation of A2B adenosine receptors protects against trauma–hemorrhagic shock-induced lung injury

Inflammation is responsible for secondary organ failure after trauma and hemorrhagic shock (T/HS). Adenosine, acting through four G protein-coupled cell surface receptors, A₁, A_2A, A_2B, and A₃, exerts a number of tissue protective and anti-inflammatory effects. The goal of the present study was to test the effect of A_2B adenosine receptor stimulation on T/HS-induced organ injury and inflammation in rats. Rats after T/HS were resuscitated with Ringer’s lactate containing the A_2B receptor agonist BAY 60–6583 or its vehicle. We found that BAY 60–6583 decreased T/HS-induced lung permeability and plasma creatine kinase levels but failed to affect T/HS-induced lung neutrophil infiltration and IκBα expression and plasma alanine aminotransferase levels. Thus, we conclude that stimulation of A_2B receptors protects against T/HS-induced lung and muscle injury.     

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

Neuroblastoma SH-SY5Y (SH) cells endogenously express A(2A) adenosine receptors and can be differentiated into a sympathetic neuronal phenotype, capable of depolarisation-dependent noradrenaline release. Using differentiated SH culture, we here explored the link between A(2A)-receptor signalling and neurotransmitter release. In response to the receptor agonist CGS21680, the cells produced cyclic AMP (cAMP), and when depolarised, they released increased amounts of noradrenaline. An A(2A)-receptor antagonist, XAC, as well as an inhibitor of cAMP-dependent protein kinase A (PKA), H89, depressed agonist-dependent release. In the presence of XAC or H89, noradrenaline release was found to be below basal values. This suggested that release facilitation also owes to constitutive receptor activity. We demonstrate that even in the absence of an agonist, the native A(2A)-receptor stimulated cAMP production, leading to the activation of PKA and enhanced noradrenaline release. Ancillary, non-cAMP-dependent effects of the receptor (i.e. phosphorylation of CREB, of Rabphilin3A) were refractory to constitutive activation. PKA-dependent facilitation of noradrenaline release was recapitulated with membrane-permeable 8-Br-cAMP; in addition to facilitation, 8-Br-cAMP caused marked inhibition of release, an effect not observed upon receptor activation. Inhibition by receptor-independent cAMP was likely due to suppression of voltagedependent calcium current (VDCC) and increased activity of Src-family kinases. Receptor-mediated release facilitation was reproduced in the presence of tetrodotoxin (blocking action potentials); hence, the signalling occurred at the active zone comprising release sites. Our findings thus support (1) presynaptic localisation of the A(2A)-receptor and (2) suggest that compartmentalised pathways transmit cAMP signalling in order to facilitate depolarisation-dependent neurotransmitter release.     

TNF-alpha upregulates the A2B adenosine receptor gene: The role of NAD(P)H oxidase 4

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-α) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A_2B adenosine receptor (A_2BAR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-α. Here, we show a regulatory loop by which TNF-α upregulates the A_2BAR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A_2BAR, and Nox inhibitors dampen the effect of TNF-α. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A_2BAR.