Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth,Neovascularization, Angiogenesis and Macrophage Infiltration

CD73 (ecto-5''-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A₁, A_2A, and A₃ in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A₁AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A₃AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.     

Human brain endothelial cells are responsive to adenosine receptor activation

The blood–brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A₁, A_2A, and A_2B AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5′-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.     

Adenosine A<Subscript>2A</Subscript> receptor and ecto-5′-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE)

Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A_2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A₁ receptors. Gain of function of the excitatory A_2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A_2A receptor and of the adenosine-generating enzyme, ecto-5′-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A_2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A_2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A_2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A_2A receptor and ecto-5′-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A_2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5′-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.     

Pharmacophore mapping and in silico screening to identify new potent leads for A2A adenosine receptor as antagonists

A_2A adenosine receptor (AR) antagonists play an important role in neurodegenerative diseases like Parkinson’s disease. A 3D-QSAR study of A_2A AR antagonists, was taken up to design best pharmacophore model. The pharmacophoric features (ADHRR) containing a hydrogen bond acceptor (A), a hydrogen bond donor (D), a hydrophobic group (H) and two aromatic rings (R), is projected as the best predictive pharmacophore model. The QSAR model was further treated as a template for in silico search of databases to identify new scaffolds. The binding patterns of the leads with A_2A AR are analysed using docking studies and novel potent ligands of A_2A AR are projected.     

Novel positive allosteric modulators of A2B adenosine receptor acting as bone mineralisation promoters

Small-molecules acting as positive allosteric modulators (PAMs) of the A_2B adenosine receptor (A_2B AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds (4-13) exhibiting different degrees of chemical similarity with three indole derivatives (1-3), which have been recently identified by us as PAMs of the A_2B AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of 4-13 in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative 9, confirmed that such a molecule behaves as PAM of the A_2B AR.     

Alterations in Adenosine Metabolism and Signaling in Patients with Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis

Background

Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated.

Methodology/Principal Findings

The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A_2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A_2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A_2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A_2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators.

Conclusions/Significance

These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A_2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.     

Synthesis,biological evaluation and molecular modelling studies of 1,3,7,8-tetrasubstituted xanthines as potent and selective A2A AR ligands with in vivo efficacy against animal model of Parkinson’s disease

In the present study, an attempt has been made to develop a new series of 1,3,7,8-tetrasubstituted xanthine based potent and selective AR ligands for the treatment of Parkinson's disease. Antagonistic interactions between dopamine and A_2A adenosine receptors serve as the basis for the development of AR antagonists as potential drug candidates for PD. All the synthesized compounds have been evaluated for their affinity toward AR subtypes using in vitro radioligand binding assays. 1,3-Dipropylxanthine 7a with a methyl substituent at N-7 position represents the most potent compound of the series and displayed highest affinity (A_2A, K_i = 0.108 µM), however incorporation of a propargyl group at 7-positon of the xanthine nucleus seems to be the most appropriate substitution to improve selectivity towards the A_2A subtype along with reasonable potency. Antiparkinsonian activity has been evaluated using perphenazine induced catatonia in rats. Most of the synthesized xanthines significantly lowered the catatonic score as compared to control and displayed antiparkinsonian effects comparable to standard drug. All the synthesized compounds were subjected to grid-based molecular docking studies to understand the key structural requirements for the development of new molecules well-endowed with intrinsic efficacy and selectivity as adenosine receptor ligands. In silico studies carried out on newly synthesized xanthines provided further support to the pharmacological results.     

Synthesis and structure–activity relationships of 2-hydrazinyladenosine derivatives as A2A adenosine receptor ligands

A series of 2-hydrazinyladenosine derivatives was synthesized and investigated in radioligand binding studies for their affinity at the adenosine receptor subtypes with the goal to obtain potent and A_2AAR selective agonists and to explore the structure–activity relationships of this class of compounds at A_2AAR. Modifications included introduction of a second sugar moiety at position 2 of adenosine to form new bis-sugar nucleosides and/or modifications of the 2-position linker in different ways. The performed modifications were found to produce compounds with relatively high A_2AAR affinity and very high selectivity toward A_2AAR. The most potent bis-sugar nucleoside was obtained with the d-galactose derivative 16 which exhibited a K_i value of 329 nM at A_2AAR with marked selectivity against the other AR subtypes. In another set of compounds, compound 3 was modified via replacement of its cyclic structure with mono- and disubstituted phenyl moieties and the resulting hydrazones 10–14 were found to have low nanomolar affinity for A_2AAR. In addition to 3, compounds 10, 11 and 13 have been identified as the most potent compounds in the present series with K_i values of 16.1, 24.4, and 12.0 nM, respectively, at rat A_2AAR. Species differences were tested and found to exist in different rates. Functional properties of the most potent compounds 10, 11, 13 and 16 were assessed showing that the compounds acted as agonists at A_2AAR.     

Limits of Ligand Selectivity from Docking to Models: In Silico Screening for A1 Adenosine Receptor Antagonists

G protein-coupled receptors (GPCRs) are attractive targets for pharmaceutical research. With the recent determination of several GPCR X-ray structures, the applicability of structure-based computational methods for ligand identification, such as docking, has increased. Yet, as only about 1% of GPCRs have a known structure, receptor homology modeling remains necessary. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A₁ adenosine receptor (A₁AR) and docked ∼2.2 M lead-like compounds. High-ranking molecules were tested on the A₁AR as well as the close homologs A_2AAR and A₃AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. These findings have implications for future screens.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

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.     

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators

The A_2B adenosine receptor (A_2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A_2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases.Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A_2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A_2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization.In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A_2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A_2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A_2B AR in the control of osteoblast differentiation, bone formation and fracture repair.     

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.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

Downregulation of A1 and A2B adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A₁ and A_2B expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5′-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A₁AR and A_2AAR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A_2B. In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A_2B and A₁ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A₁ and A_2BARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.     

Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A₁ and A_2A receptors (A₁AR and A_2AAR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A₁AR and A_2AAR, respectively. To proof the feasibility of MS binding on the A₁AR and A_2AAR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9477-0) contains supplementary material, which is available to authorized users.     

Adenosine A(2B) receptor mediates an increase on VEGF-A production in rat kidney glomeruli

Up-regulation of the glomerular expression and the activity of vascular endothelial growth factor-A (VEGF) have been identified as an early pathogenic event for the progression of diabetic nephropathy. Currently, however the mediators are not yet clearly recognized. In this study we identified all four adenosine receptor (AR) subtypes, i.e. A₁, A_2A, A_2B and A₃ in isolated rat kidney glomeruli. We localized the expression of A_2BAR in podocytes, the primary VEGF producing cells. The ex vivo treatment of kidney glomeruli with adenosine or a general AR agonist NECA, increases VEGF protein content. In addition, NECA treatment elicits VEGF release. These effects were blocked by the A_2BAR selective antagonist MRS1754 supplementation. Furthermore, we showed that A_2BAR activation was necessary to promote a higher expression of VEGF in kidney glomeruli upon exposure to high d-glucose concentration, a pathogenic condition like those observed in diabetic nephropathy.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.