Human Adenosine Research Articles

Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing

As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.

Relationship of human microvascular endothelial cell activation and adenosine level under the hypoxia

Background Retinal hypoxia is one of primary causes of retinal neovascularization, and its mechanism is research hot topic.Studies showed that hypoxia stimulates the activation of many trancripation factors and vascular endothelial cells, which leads to angiogenesis.Besides to the vascular endothelial growth factor, the effect of adenosine on angiogenesis is increasingly concerned. Objective This study was to invastigate the relationship of biological behaviour of human microvascular endothelial cells (HMEC-1) under the hypoxia and adenosine, and to explore the effect of adenosine on angiogenesis under hypoxia. Methods HMEC-1 cell line was cultured in vitro, and the cells were divided into normoxia group and hypoxia group.The cells in the normoxia group were cultured under the 5%CO2 environment, and those in the hypoxia group were cultured under the 1% O2, 94%N2 and 5%CO2 environment.The proliferation ability and percentage of the cells were assayed by cell counting kit-8 (CCK8) and EDU.The migration number and invasive number of the cells were detected by transwell chamber.The expressions of CD39 and CD73 proteins in the cells were tested by Western blot and immunofluorescence technique, and the level of adenosine was measured by high performance liquid chromatography (HPLC). Results The proliferation values (absobancy) were 0.715±0.067 and 0.821±0.056 in the normoxia group and the hypoxia group in 12 hours after culture, and those in 24 hours were 0.946±0.028 and 0.998±0.028, showing significant increase in the hypoxia group compared with the normoxia group (t12h= 3.805, t24h= 3.222, all at P<0.01). The precentage of the proliferation in the hypoxia group was evidently higher than that in the normoxia group (t=-6.868, P<0.01). The number of the cell migration and invasion in 24 hours after culture was 185.3±10.594 and 74.2±10.741 respectively in the normoxia group, and that in the hypoxia group was 300.7±22.853 and 107.5±7.007, with significant differences between the two groups (t=-12.124, -6.367, both at P<0.01). The expression levels of CD39 and CD73 proteins in the hypoxia group were significantly higher than those in the normoxia group in both12 hours and 24 hours after culture(all at P<0.05), and the adenosine content in the cells is gradually increased in 2, 6, 12, 24 and 36 hours after culture, with the highest content in 36 hours.The adenosine content was significantly higher in the hypoxia group than in the normoxia group at various time points (t2h=2.469, P=0.017; t6h=5.442, P<0.001; t12h=3.841, P<0.001; t24h=4.458, P<0.001; t36h=2.757, P=0.008; t48h=3.319, P=0.002). Conclusions Compared with the normoxia group, the proliferation, migration and invasion abilities of HMEC-1 are stronger, meanwhile, the expression of CD39 and CD73 as well as the adenosine level in the cells are all increased under the hypoxic condition.It is suggested that the activation of human microvascular endothelial cells might be significantly related to the level of adenosine and its key enzymes. Key words: Neovascularization; Retina; Microvascular endothelial cells; Human; Angiogenesis; adenosine

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15–24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.

Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein.

G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR-G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are challenging targets for structural studies due to their conformationally dynamic and inherently transient nature. We recently developed an engineered G protein, mini-Gs, which addressed these problems and allowed the formation of a stable GPCR-G protein complex. Mini-Gs facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its G protein-bound conformation at 3.4 Å resolution. Here, we describe a step by step protocol for the expression and purification of A2AR, and crystallisation of the A2AR-mini-Gs complex.

Expression and Purification of Mini G Proteins from Escherichia coli.

Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

A covalent antagonist for the human adenosine A2A receptor

The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.

Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective adenosine A1 receptor antagonists

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A1 receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a subnanomolar affinity but limited selectivity over the A2A subtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA1AR by introducing substituents on the N2-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (Ki(hA1AR) = 7.7 nM) and selective (Ki(hA2AAR) = 1389 nM) antagonist at the human adenosine A1 receptor. Computational docking was effected at the A1 and A2A subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.

Human adenosine deaminases ADA1 and ADA2 bind to different subsets of immune cells.

At sites of inflammation and tumor growth, the local concentration of extracellular adenosine rapidly increases and plays a role in controlling the immune responses of nearby cells. Adenosine deaminases ADA1 and ADA2 (ADAs) decrease the level of adenosine by converting it to inosine, which serves as a negative feedback mechanism. Mutations in the genes encoding ADAs lead to impaired immune function, which suggests a crucial role for ADAs in immune system regulation. It is not clear why humans and other mammals possess two enzymes with adenosine deaminase activity. Here, we found that ADA2 binds to neutrophils, monocytes, NK cells and B cells that do not express CD26, a receptor for ADA1. Moreover, the analysis of CD4+ T-cell subset revealed that ADA2 specifically binds to regulatory T cells expressing CD39 and lacking the receptor for ADA1. Also, it was found that ADA1 binds to CD16- monocytes, while CD16+ monocytes preferably bind ADA2. A study of the blood samples from ADA2-deficient patients showed a dramatic reduction in the number of lymphocyte subsets and an increased concentration of TNF-α in plasma. Our results suggest the existence of a new mechanism, where the activation and survival of immune cells is regulated through the activities of ADA2 or ADA1 anchored to the cell surface.

Polydopamine Nanotubes as an Effective Fluorescent Quencher for Highly Sensitive and Selective Detection of Biomolecules Assisted with Exonuclease III Amplification.

In this work, the effective fluorescence quenching ability of polydopamine nanotubes (PDANTs) toward various fluorescent dyes was studied and further applied to fluorescent biosensing for the first time. The PDANTs could quench the fluorophores with different emission frequencies, aminomethylcoumarin acetate (AMCA), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), and Cy5. All the quenching efficiencies reached to more than 97%. Taking advantage of PDANTs' different affinities toward ssDNA and dsDNA and utilizing the complex of FAM-labeled ssDNA and PDANTs as a sensing platform, we achieved highly sensitive and selective detection of human immunodeficiency virus (HIV) DNA and adenosine triphosphate (ATP) assisted with Exonuclease III amplification. The limits of detection (LODs) of HIV DNA and ATP reached to 3.5 pM and 150 nM, respectively, which were all lower than that of previous nanoquenchers with Exo III amplification, and the platform also presented good applicability in biological samples. Fluorescent sensing applications of this nanotube enlightened other targets detection based upon it and enriched the building blocks of fluorescent sensing platforms. This polydopamine nanotube also possesses excellent biocompatibility and biodegradability, which is suitable for future drug delivery, cell imaging, and other biological applications.

Glutathione S-transferase ϴ-subunit as a phenotypic suppressor of pmr1Δ strain, the Kluyveromyces lactis model for Hailey-Hailey disease

BackgroundHailey–Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1, the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump. MethodsIn this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1, the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells. ResultsWe have identified the Glutathione S-transferase ϴ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δmutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca2+) homeostasis by decreasing the high cytosolic Ca2+ levels in pmr1Δstrain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart (GSTT1/M1) was observed in lesion-derived keratinocytes from HHD patients. ConclusionsThese data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease. General significanceKluyveromyces lactis can be considered a good model to study the molecular basis of this pathology.

Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.

Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens.

Similarities and differences in affinity and binding modes of tricyclic pyrimido- and pyrazinoxanthines at human and rat adenosine receptors

A new series of 32 pyrimido- and 5 tetrahydropyrazino[2,1-f]purinediones was obtained and evaluated for their adenosine receptors (ARs) affinities. The 1,3-dibutyl derivative of 9-(4-(2-(dimethylamino)ethoxy)phenyl)-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)-dione was found to be the most potent A1 AR antagonist of the present series, showing selectivity over the other AR subtypes. The structure–activity for the obtained purinediones was established. Docking experiments of the investigated library to homology models of the human and rat A1 and A2A ARs allowed to compare the expected binding modes for selected compounds. The detailed analysis of binding cavities within individual AR subtypes indicated small but significant structural variations that may underlie the observed differences in binding affinities of purinediones at particular subtypes and species.

South (S)- and North (N)-Methanocarba-7-Deazaadenosine Analogues as Inhibitors of Human Adenosine Kinase.

Adenosine kinase (AdK) inhibitors raise endogenous adenosine levels, particularly in disease states, and have potential for treatment of seizures, neurodegeneration, and inflammation. On the basis of the South (S) ribose conformation and molecular dynamics (MD) analysis of nucleoside inhibitors bound in AdK X-ray crystallographic structures, (S)- and North (N)-methanocarba (bicyclo[3.1.0]hexane) derivatives of known inhibitors were prepared and compared as human (h) AdK inhibitors. 5'-Hydroxy (34, MRS4202 (S); 55, MRS4380 (N)) and 5'-deoxy 38a (MRS4203 (S)) analogues, containing 7- and N(6)-NH phenyl groups in 7-deazaadenine, robustly inhibited AdK activity (IC50 ∼ 100 nM), while the 5'-hydroxy derivative 30 lacking the phenyl substituents was weak. Docking in the hAdK X-ray structure and MD simulation suggested a mode of binding similar to 5'-deoxy-5-iodotubercidin and other known inhibitors. Thus, a structure-based design approach for further potency enhancement is possible. The potent AdK inhibitors in this study are ready to be further tested in animal models of epilepsy.

Synthesis and Evaluation of a New Series of 8-(2-Nitroaryl)Xanthines as Adenosine Receptor Ligands.

Preclinical Research A new series of 1,3-dimethylxanthine derivatives bearing 8-(2-nitroaryl) residue was synthesized and evaluated for affinity for recombinant human adenosine receptors subtypes. Nitrate esters of 7-substituted-1,3-dimethyl-8-phenylxanthines were also synthesized and tested. Introducing a nitro substituent at the 2-position of the 8-substituted phenyl ring resulted in generally low affinity for adenosine receptors (ARs), selectivity toward the A2A subtype was enhanced in some of the compounds. 8-(4-Cyclopentyloxy-5-methoxy-2-nitrophenyl)-1,3-dimethylxanthine (9e) proved to be a potent compound among the 2-nitrophenyl substituted xanthines exhibiting a Ki = 1 μM at human A2A ARs with at least 30 fold selectivity versus human A1 and A2B ARs. Replacement of 8-chloropropoxy phenyl with 8-nitrooxypropoxy phenyl resulted in a negligible change in binding affinity of the 8-substituted xanthines for various AR subtypes. Drug Dev Res 77 : 241-250, 2016. © 2016 Wiley Periodicals, Inc.

4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives as novel non-nucleoside agonists for the adenosine A1 receptor.

Three 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives (4-6) were investigated as potential non-nucleoside agonists at human adenosine receptors (ARs). When tested in competition binding experiments, these compounds exhibited low micromolar affinity (Ki values comprised between 1.2 and 1.9μm) for the A1 AR and no appreciable affinity for the A2A and A3 ARs. Evaluation of their efficacy profiles by measurement of intracellular cAMP levels revealed that 4 and 5 behave as non-nucleoside agonists of the A1 AR with EC50 values of 0.47 and 0.87μm, respectively. No clear concentration-response curves could be instead obtained for 6, probably because this compound modulates one or more additional targets, thus masking the putative effects exerted by its activation of A1 AR. The three compounds were not able to modulate A2B AR-mediated cAMP accumulation induced by the non-selective AR agonist NECA, thus demonstrating no affinity toward this receptor.

Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

Resistance to chemotherapy is associated with altered glucose metabolism in acute myeloid leukemia.

Altered glucose metabolism has been described as a cause of chemoresistance in multiple tumor types. The present study aimed to identify the expression profile of glucose metabolism in drug-resistant acute myeloid leukemia (AML) cells and provide potential strategies for the treatment of drug-resistant AML. Bone marrow and serum samples were obtained from patients with AML that were newly diagnosed or had relapsed. The messenger RNA expression of hypoxia inducible factor (HIF)-1α, glucose transporter (GLUT)1, and hexokinase-II was measured by quantitative polymerase chain reaction. The levels of LDH and β subunit of human F1-F0 adenosine triphosphate synthase (β-F1-ATPase) were detected by enzyme-linked immunosorbent and western blot assays. The HL-60 and HL-60/ADR cell lines were used to evaluate glycolytic activity and effect of glycolysis inhibition on cellular proliferation and apoptosis. Drug-resistant HL-60/ADR cells exhibited a significantly increased level of glycolysis compared with the drug-sensitive HL-60 cell line. The expression of HIF-1α, hexokinase-II, GLUT1 and LDH were increased in AML patients with no remission (NR), compared to healthy control individuals and patients with complete remission (CR) and partial remission. The expression of β-F1-ATPase in patients with NR was decreased compared with the expression in the CR group. Treatment of HL-60/ADR cells with 2-deoxy-D-glucose or 3-bromopyruvate increased in vitro sensitivity to Adriamycin (ADR), while treatment of HL-60 cells did not affect drug cytotoxicity. Subsequent to treatment for 24 h, apoptosis in these two cell lines showed no significant difference. However, glycolytic inhibitors in combination with ADR increased cellular necrosis. These findings indicate that increased glycolysis and low efficiency of oxidative phosphorylation may contribute to drug resistance. Targeting glycolysis is a viable strategy for modulating chemoresistance in AML.

5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site.

Human adenosine deaminases control the immune cell responses to activation signals by reducing extracellular adenosine concentration

Abstract Adenosine rapidly accumulates in the sites of inflammation and tumor growth. It binds to adenosine receptors expressed on the cell surface of immune cells and induces either suppression or activation of inflammatory responses to pathogens. In humans the level of extracellular adenosine is regulated by two adenosine deaminases ADA1 and ADA2. Decrease in ADAs concentration due to genetic defects in the ADA genes leads to serious perturbation in the immune system function while increase in ADA activity associates with numerous immune diseases and cancers. The immune responses to extracellular adenosine have largely been studied using pharmacological approach where non-hydrolysable adenosine receptors agonists substitute adenosine to form the activated state of adenosine receptors. On contrary, adenosine receptors bound to adenosine receptor antagonists mimic inactivated state of adenosine receptors. Here, the effect of adenosine receptor agonists and antagonists on the monocytes function as well as and T helper cell proliferation and differentiation was compared with the effect of adenosine and adenosine deaminases. It was demonstrated that adenosine deaminases control the immune cells responses to activation signals by reducing the concentration of extracellular adenosine and that the cells sensitivity to adenosine greatly depends on the type of the cell activation. Therefore, our data suggests that ADAs could be considered as new drug candidates for the treatment of immune disorders and cancers.

Exploring the 2- and 5-positions of the pyrazolo[4,3-d]pyrimidin-7-amino scaffold to target human A1 and A2A adenosine receptors

A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1–31) were synthesized to evaluate some structural modifications at the 2- and 5-positions aimed at shifting affinity towards the human (h) A2A adenosine receptor (AR) or both hA2A and hA1 ARs. The most active compounds were those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a substituted-benzyl group at position 2. Several of these derivatives (22–31) displayed nanomolar affinity for the hA2A AR (Ki=3.62–57nM) and slightly lower for the hA1 ARs, thus showing different degrees (3–22 fold) of hA2A versus hA1 selectivity. In particular, the 2-(2-methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 25 possessed the highest hA2A and hA1 AR affinities (Ki=3.62nM and 18nM, respectively) and behaved as potent antagonist at both these receptors (cAMP assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA2A AR (Ki=5.26nM) and was 22-fold selective versus the hA1 subtype. Molecular docking investigations performed at the hA2A AR crystal structure and at a homology model of the hA1 AR allowed us to represent the hypothetical binding mode of our derivatives and to rationalize the observed SARs.