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Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A 2B receptor (A 2B AR). We have synthetically implemented the recently reported N -acyl- N -alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A 2B AR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A 2B AR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A 2B AR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.

The adenosine subfamily G protein-coupled receptors A 2A R and A 2B R have been identified as promising cancer immunotherapy candidates. One of the A 2A R/A 2B R dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A 2A R complexed with AB928 and a selective A 2A R antagonist 2-118. The structures revealed a common binding mode on A 2A R, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A 2A R and A 2B R molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A 2B R. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A 2B R pocket, while 2-118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A 2B R and A 2A R. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A 2A R/A 2B R for cancer therapy., (© 2024. Science China Press.)

Introduction: A 2B adenosine receptor (A 2B AR) plays a crucial role in pathophysiologic conditions associated with high adenosine release, typical of airway inflammatory pathologies, gastrointestinal disorders, cancer, asthma, type 2 diabetes, and atherosclerosis. In some pathologies, simultaneous inactivation of A 2A and A 2B ARs is desirable to have a synergism of action that leads to a greater efficacy of the pharmacological treatment and less side effects due to the dose of drug administered. In this context, it is strongly required to identify molecules capable of selectively antagonizing A 2B AR or A 2A /A 2B ARs., Areas Covered: The review provides a summary of patents, published from 2016 to present, on chemicals and their clinical use. In this paper, information on the biological activity of representative structures of recently developed A 2B or A 2A /A 2B receptor ligands is reported., Expert Opinion: Among the four P1 receptors, A 2B AR is the most inscrutable and the least studied until a few years ago, but its involvement in various inflammatory pathologies has recently made it a pharmacological target of high interest. Many efforts by the academy and pharmaceutical companies have been made to discover potential A 2B AR and A 2A /A 2B ARs drugs. Although several compounds have been synthesized only a few molecules have entered clinical trials.

Sickle cell disease (SCD) is a disease resulting from mutation in the globin portion of hemoglobin caused by the replacement of adenine for thymine in the codon of the β globin gene. In Brazil, SCD affects about 0.3% of the black and Caucasian population. Until now, there is no specific treatment and the available drugs have several serious adverse effects which makes the search for new drugs an emergently need. The use of computational techniques can accelerate the drug development process by prioritization of molecules with affinity against essential targets. Adenosine A2b receptor (rA2b) has been studied in SCD due to its relationship with red blood cells concentration of 2,3-diphosphoglycerate which reduces the hemoglobin affinity for oxygen (O 2 ), facilitating its availability for the tissues. Then, development of rA2b antagonists could be helpful for the treatment of SCD. However, there is still no 3D structure of rA2b and to overcome this limitation, homology modeling should be applied. In this scenario, this study aims to build a suitable 3D model of rA2b by SWISS MODEL and to evaluate the structural aspects of rA2b with known antagonists that may be useful for the identification of new potential antagonists by molecular dynamics on a lipid bilayer environment using GROMACS 5.1.4. The complexes with antagonists ZINC223070016 and ZINC17974526 interacted with key residues by hydrophobic contacts and hydrogen bonds which stabilized them at the rA2b binding site. This intermolecular profile can contribute to the development of more potent rA2b antagonists. Communicated by Ramaswamy H. Sarma.

Background: Spinal cord injury (SCI) is an inflammatory condition, and excessive adenosine triphosphate (ATP) is released into the extracellular space, which can be catabolized into adenosine by CD73. Extracellular vesicles have been designed as nano drug carriers in many diseases. However, their impacts on delivery of CD73 after SCI are not yet known. We aimed to construct CD73 modified extracellular vesicles and explore the anti-inflammatory effects after SCI., Methods: CD73 engineered extracellular vesicles (CD73+ hucMSC-EVs) were firstly established, which were derived from human umbilical cord mesenchymal stem cells (hucMSCs) transduced by lentiviral vectors to upregulate the expression of CD73. Effects of CD73+ hucMSC-EVs on hydrolyzing ATP into adenosine were detected. The polarization of M2/M1 was verified by immunofluorescence. Furthermore, A2aR and A 2b R inhibitors and A2bR knockdown cells were used to investigate the activated adenosine receptor. Biomarkers of microglia and levels of cAMP/PKA were also detected. Repetitively in vivo study, morphology staining, flow cytometry, cytokine analysis, and ELISA assay, were also applied for verifications., Results: CD73+ hucMSC-EVs reduced concentration of ATP and promoted the level of adenosine. In vitro experiments, CD73+ hucMSC-EVs increased macrophages/microglia M2:M1 polarization, activated adenosine 2b receptor (A2bR), and then promoted cAMP/PKA signaling pathway. In mice using model of thoracic spinal cord contusion injury, CD73+ hucMSC-EVs improved the functional recovery after SCI through decreasing the content of ATP in cerebrospinal fluid and improving the polarization from M1 to M2 phenotype. Thus, the cascaded pro-inflammatory cytokines were downregulated, such as TNF-α, IL-1β, and IL-6, while the anti-inflammatory cytokines were upregulated, such as IL-10 and IL-4., Conclusions: CD73+ hucMSC-EVs ameliorated inflammation after spinal cord injury by reducing extracellular ATP, promoting A2bR/cAMP/PKA pathway and M2/M1 polarization. CD73+ hucMSC-EVs might be promising nano drugs for clinical application in SCI therapy., (© 2021. The Author(s).)

The four adenosine receptors (ARs) A 1 AR, A 2A AR, A 2B AR , and A 3 AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A 2B AR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A 2A and A 2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A 2B AR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V250 6.51 in A 2B AR, which is a leucine in all other ARs including the closely related A 2A AR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V 6.51 mutant A 2A AR receptor. Taken together, this study provides further insights in the binding mode of these A 2B AR antagonists, paving the way for future ligand optimization.

All four of the adenosine receptor (AR) subtypes mediate pain and have been targeted by pharmacologists to generate new therapeutics for chronic pain. The vanilloid phytochemicals, which include curcumin, capsaicin, and gingerol, have been shown to alleviate pain. However, there is little to no literature on the interaction of vanilloid phytochemicals with ARs. In this study, photochemical methods were used to generate a novel isomer of curcumin (cis-trans curcumin or CTCUR), and the interactions of both curcumin and CTCUR with the two G s -linked AR subtypes were studied. Competitive binding assays, docking analysis, and confocal fluorescence microscopy were performed to measure binding affinity; cell survival assays were used to measure toxicity; and cAMP assays were performed to measure receptor activation. Competitive binding results indicated that CTCUR binds to both AR A 2A and AR A 2B with K i values of 5 μM and 7 μM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A 2B and A 2A . Cell survival results show that CTCUR and CUR are nontoxic at the tested concentrations in these cell lines. Overall, our results suggest that vanilloid phytochemicals may be slightly modified to increase interaction with G s -ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory impairment. Adenosinergic receptors are considered as a potential alternative in the management of several neurodegenerative disorders. However, there is no information available on the role of A2b receptor in the pathophysiology of AD. Therefore, the effect of Aβ on the level of expression of A2b receptor was investigated in discrete memory-sensitive mouse brain regions. Aβ (1-42) was injected intracerebroventricularly to healthy male mouse to induce AD-like behavioral manifestations on Day-1 (D-1) of the experimental protocol. The animals were subjected to the Morris water maze (MWM) test on D-14 to D-18. On D-18, the animals were subjected to the Y-maze test after 30 min lag to the MWM paradigm. Aβ significantly attenuated the spatial working memory in MWM and Y-maze tests. In addition, Aβ significantly increased cholinergic dysfunction in terms of decrease in the activity of ChAT and ACh level and increase in the AChE activity in the hippocampus, pre-frontal cortex and amygdala of AD-like animals. Further, there was a significant increase in the extent of apoptosis in the selected mouse brain regions. Moreover, Aβ caused a substantial reduction in the mitochondrial function, integrity and bioenergetics in all the mouse brain regions. Furthermore, there was a significant decrease in the level of expression of A2b receptors in the selected brain regions of the rodents. Hence, it can be assumed that A2b receptor downregulation could be another therapeutic target in the management of AD.

Currently, the most promising therapeutic modality for cancer treatment is the blockade of immune checkpoint pathways, which has revolutionized cancer therapy in the past 15 years. Strategies targeting and modulating adenosine A 2A receptor (A 2A R), an emerging alternative immune checkpoint, have shown the potential to produce significant therapeutic effects. In this review, we describe the immunosuppressive activities of A 2A R and A 2B R in the tumor microenvironment (TME), followed by a summary and discussion of the structure-activity relationship (SAR) of the A 2A R (and dual A 2A R/A 2B R) antagonists that have been experimentally confirmed to exert oncoimmunological effects. This review also provides an update on the compounds under clinical evaluation and insights into the ligand binding modes of the receptor.

Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABA A ) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A 2B adenosine receptor (A 2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.

A 2A and A 2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A 2A ARs are activated by low (nanomolar) adenosine concentrations, while A 2B ARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A 2A AR for that of the A 2B AR. The resulting chimeric A 2A (ECL2-A 2B )AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A 2A AR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A 2A (ECL2-A 2B )AR mutant displaying similarly low potency as for the wt A 2B AR. Supervised molecular dynamics simulation studies predicted a meta-binding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We report the identification of two subsets of fluorinated nonxanthine A 2B adenosine receptor antagonists. The novel derivatives explore the effect of fluorination at different positions of two pyrimidine-based scaffolds. The most interesting ligands combine excellent hA 2B affinity ( K i < 15 nM) and remarkable subtype selectivity. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The compounds were designed on the basis of previous molecular models of the stereoselective binding of the parent scaffolds to the hA 2B receptor, and we herein provide refinement of such models with the fluorinated compounds, which allows the explanation of the spurious effects of the fluorination at the different positions explored. These models are importantly confirmed by a synergistic study combining chiral HPLC, circular dichroism, diastereoselective synthesis, molecular modeling, and X-ray crystallography, providing experimental evidence toward the stereospecific interaction between optimized trifluorinated stereoisomers and the hA 2B receptor.

Adenosine receptors (ARs) are classified as A 1 , A 2A , A 2B , and A 3 subtypes belonging to the superfamily of G-protein-coupled receptors (GPCRs). Several molecular modeling approaches have been developed for A 2B AR and its antagonists, from the construction of a homology model, molecular docking, molecular dynamics (MD) simulations, and 3D quantitative structure-activity relationship (QSAR) modeling to pharmacophore modeling, each of which has different objectives and outcomes. In this review, we provide a systematic outline of advances in molecular modeling approaches towards A 2B AR for deducing its structure and interactions with various types of antagonist. The information, methods and perspectives presented here provides impetus for medicinal chemists to discover potential ligands that can bind selectively with higher affinity to A 2B AR., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Adenosine is a ubiquitous endogenous nucleoside that controls numerous physiological functions via interacting with its specific G-coupled receptors. Activation of adenosine receptors (AdoRs), particularly A 2B AdoRs promotes the release of inflammatory cytokines; reduces vascular permeabilization and induces angiogenesis, thereby making A 2B AdoR becomes a potentially pharmacological target for drug development. Presently, for investigating the structural determinants of 164 xanthine derivatives as A 2B AdoR antagonists, we performed an in silico study integrating with 3D-QSAR, docking and molecular dynamics (MD) simulation. The obtained optimal model shows strong predictability ( Q 2  = 0.647, R 2 ncv  = 0.955, and R 2 pred  = 0.848). Additionally, to explore the binding mode of the ligand with A 2B AdoR and to understand their binding mechanism, docking analysis, MD simulations (20 ns), and the calculation of binding free energy were also carried out. Finally, the structural determinants of these xanthine derivatives were identified and a total of 20 novel A 2B AdoR antagonists with improved potency were computationally designed, and their synthetic feasibility and selectivity were also evaluated. The information derived from the present study offers a better appreciation for exploring the interaction mechanism of the ligand with A 2B AdoR, which could be helpful for designing novel potent A 2B AdoR antagonists. Communicated by Ramaswamy H. Sarma.

The A 2B adenosine receptor (A 2B AR) was proposed as a novel target for the (immuno)therapy of cancer since A 2B AR blockade results in antiproliferative, antiangiogenic, antimetastatic, and immunostimulatory effects. In this study, we explored the structure-activity relationships of xanthin-8-yl-benzenesulfonamides mainly by introducing a variety of linkers and substituents attached to the sulfonamide residue. A new, convergent strategy was established, which facilitated the synthesis of the target compounds. Many of the new compounds exhibited subnanomolar affinity for the A 2B AR combined with high selectivity. Functional groups were introduced, which will allow the attachment of dyes and other reporter groups. 8-(4-((4-(4-Bromophenyl)piperazin-1-yl)sulfonyl)phenyl)-1-propylxanthine (34, PSB-1901) was the most potent A 2B -antagonist ( K i 0.0835 nM, K B 0.0598 nM, human A 2B AR) with >10 000-fold selectivity versus all other AR subtypes. It was similarly potent and selective at the mouse A 2B AR, making it a promising tool for preclinical studies. Computational studies predicted halogen bonding to contribute to the outstanding potency of 34.

Background/aims: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model., Methods: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity., Results: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La 3+ , both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A 2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate., Conclusion: The Calu-3 cell model shows that stimulation of the A 2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

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 N 1 (1,2) or N 10 (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A 1 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 A 1 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 A 1 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 A 1 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A selective agonist radioligand for A 2B adenosine receptors (A 2B ARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A 2B AR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [ 3 H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A 2B AR agonist [ 3 H]NECA for its potential to label A 2B ARs. [ 3 H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A 2B ARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [ 3 H]NECA than versus the A 2B -antagonist radioligand [ 3 H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A 2B AR agonist, it displayed higher affinity versus [ 3 H]PSB-603 than versus [ 3 H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A 2B AR. In conclusion, [ 3 H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A 2B AR conformation.

On the basis of a pyrazine core structure, three new adenosine A 2B receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for 18 F-labeling. Compound 7a was docked into a homology model of the A 2B receptor based on X-ray structures of the related A 2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A 1 receptor, 7a was radiolabeled as the most suitable candidate (K i (A 2B ) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [ 18 F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [ 18 F]7a in mice revealed the formation of a blood-brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fluorescent ligands represent powerful tools for biological studies and are considered attractive alternatives to radioligands. In this study, we developed fluorescent antagonists for A 2B adenosine receptors (A 2B ARs), which are targeted by antiasthmatic xanthines and were proposed as novel targets in immuno-oncology. Our approach was to merge a small borondipyrromethene (BODIPY) derivative with the pharmacophore of 8-substituted xanthine derivatives. On the basis of the design, synthesis, and evaluation of model compounds, several fluorescent ligands were synthesized. Compound 29 (PSB-12105), which displayed high affinity for human, rat, and mouse A 2B ARs ( K i = 0.2-2 nM) and high selectivity for this AR subtype, was selected for further studies. A homology model of the human A 2B AR was generated, and docking studies were performed. Moreover, 29 allowed us to establish a homogeneous receptor-ligand binding assay using flow cytometry. These compounds constitute the first potent, selective fluorescent A 2B AR ligands and are anticipated to be useful for a variety of applications.

Background/aims: Diabetes mellitus (DM) has become an increasingly epidemic metabolic disease. Vascular endothelial cells play a key role in developing the cardiovascular complications of DM. The A2B receptor is expressed in vascular endothelial cells, and may help regulate the function of endothelial cells. The aim of this study was to investigate the protective effects of oxymatrine (OMT) on human umbilical vein endothelial cells (HUVECs) from high glucose-induced cytotoxicity., Methods: Homology modeling and molecular docking analysis were used to detect the binding sites between the adenosine A2B receptor and OMT. HUVECs were cultured with control (5.5 mM) or elevated glucose (22.2 mM) in the presence or absence of 3 µM OMT or A2B siRNA for 3 days. The MTS cell viability assay was used to measure the toxicity of high glucose on HUVECs and the protective effect of OMT or A2B siRNA. The expression of the adenosine A2B receptor and CCL5 in HUVECs was detected with real-time quantitative PCR (qPCR) and Western blotting methods in each group. Levels of IL-1β and TNF-α were measured using an enzyme-linked immunosorbent assay (ELISA) kit, and the concentration of NO was detected with the nitrate reductase method. Monocyte chemotactic activity in each group was detected using Transwell chambers. Furthermore, the phosphorylation of p38 and ERK1/2 in each group was observed through the Western blotting method., Results: Homology modeling and molecular docking analysis showed that OMT contains well-fitted binding sites to the A2B receptor. After chronic culture at high glucose, the rate of cell viability was significantly lower than that of the control group. After co-treatment with OMT or A2B siRNA, cell viability was significantly increased compared with the high-glucose group. The results from real-time quantitative RT-PCR (qRT-PCR) and Western blotting indicated that high glucose could increase the expression of A2B receptors in HUVECs, an effect that was inhibited by OMT. In addition, the results revealed that the expression of CCL5, IL-1β and TNF-α was increased in the high-glucose group, and that the NO produced by HUVECs decreased due to hyperglycemia; however, co-culture with OMT or A2B siRNA abolished these effects. Meanwhile, the chemotaxis activity of monocytes to HUVECs cultured in high-glucose medium was enhanced 2.59-fold compared to the control cells. However, the inflammatory reactions in HUVECs were completely relieved by co-treatment with OMT or A2B siRNA. Moreover, the phosphorylation of p38 and ERK1/2 in HUVECs in the high-glucose group was significantly higher than that of the control group; these effects were reversed after co-treatment with OMT or A2B siRNA., Conclusion: OMT may protect the HUVECs from high glucose-induced cytotoxicity through inhibitting the expression of A2B receptor and inflammatory factors as well as decreasing the phosphorylation of p38 and ERK1/2., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through activation of A 2B adenosine receptor (A 2B AdoR). Selective antagonists have been shown to attenuate airway reactivity and improve inflammatory conditions in pre-clinical studies. Hence, the identification of novel, potent and selective A 2B AdoR antagonist may be beneficial for the potential treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD). Towards this effort, we explored several prop-2-ynylated C8-aryl or heteroaryl substitutions on xanthine chemotype and found that 1-prop-2-ynyl-1H-pyrazol-4-yl moiety was better tolerated at the C8 position. Compound 59, exhibited binding affinity (K i ) of 62 nM but was non-selective for A 2B AdoR over other AdoRs. Incorporation of substituted phenyl on the terminal acetylene increased the binding affinity (K i ) significantly to <10 nM. Various substitutions on terminal phenyl group and different alkyl substitutions on N-1 and N-3 were explored to improve the potency, selectivity for A 2B AdoR and the solubility. In general, compounds with meta-substituted phenyl provided better selectivity for A 2B AdoR compared to that of para-substituted analogs. Substitutions such as basic amines like pyrrolidine, piperidine, piperazine or cycloalkyls with polar group were tried on terminal acetylene, keeping in mind the poor solubility of xanthine analogs in general. However, these substitutions led to a decrease in affinity compared to compound 59. Subsequent SAR optimization resulted in identification of compound 46 with high human A 2B AdoR affinity (K i  = 13 nM), selectivity against other AdoR subtypes and with good pharmacokinetic properties. It was found to be a potent functional A 2B AdoR antagonist with a K i of 8 nM in cAMP assay in hA 2B -HEK293 cells and an IC 50 of 107 nM in IL6 assay in NIH-3T3 cells. Docking study was performed to rationalize the observed affinity data. Structure-activity relationship (SAR) studies also led to identification of compound 36 as a potent A 2B AdoR antagonist with K i of 1.8 nM in cAMP assay and good aqueous solubility of 529 μM at neutral pH. Compound 46 was further tested for in vivo efficacy and found to be efficacious in ovalbumin-induced allergic asthma model in mice., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

A novel family of structurally simple, potent, and selective nonxanthine A 2B AR ligands was identified, and its antagonistic behavior confirmed through functional experiments. The reported alkyl 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahy-dropyrimidine-5-carboxylates (16) were designed by bioisosteric replacement of the carbonyl group at position 2 in a series of 3,4-dihydropyrimidin-2-ones. The scaffold (16) documented herein contains a chiral center at the heterocycle. Accordingly, the most attractive ligand of the series [(±)16b, K i = 24.3 nM] was resolved into its two enantiomers by chiral HPLC, and the absolute configuration was established by circular dichroism. The biological evaluation of both enantiomers demonstrated enantiospecific recognition at A 2B AR, with the (S)-16b enantiomer retaining all the affinity (K i = 15.1 nM), as predicted earlier by molecular modeling. This constitutes the first example of enantiospecific recognition at the A 2B adenosine receptor and opens new possibilities in ligand design for this receptor.

We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A2BAR (A2B adenosine receptor) has been implicated in several physiological conditions, such as allergic or inflammatory disorders, vasodilation, cell growth and epithelial electrolyte secretion. For mediating the protein-protein interactions of A2BAR, the receptor's C-terminus is recognized to be crucial. In the present study, we unexpectedly found that two point mutations in the A2BAR C-terminus (F297A and R298A) drastically impaired the expression of A2BAR protein by accelerating its degradation. Thus we tested the hypothesis that these two point mutations disrupt A2BAR's interaction with a protein essential for A2BAR stability. Our results show that both mutations disrupted the interaction of A2BAR with actinin-1, an actin-associated protein. Furthermore, actinin-1 binding stabilized the global and cell-surface expression of A2BAR. By contrast, actinin-4, another non-muscle actinin isoform, did not bind to A2BAR. Thus our findings reveal a previously unidentified regulatory mechanism of A2BAR abundance., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

The adenosine receptor subfamily includes four subtypes: the A1, A2A, A2B and A3 receptors, which all belong to the superfamily of G protein-coupled receptors (GPCRs). The adenosine A2B receptor is the least investigated of the adenosine receptors, and the molecular mechanisms of its activation have hardly been explored. We used a single-GPCR-one-G protein yeast screening method in combination with mutagenesis studies, molecular modeling and bio-informatics to investigate the importance of the different amino acid residues of the NPxxY(x)6F motif and helix 8 in the human adenosine A2B receptor (hA2BR) activation. A scanning mutagenesis protocol was employed, yielding 11 single mutations and one double mutation of the NPxxY(x)6F motif and 16 single mutations of helix 8. The amino acid residues P287(7.50), Y290(7.53), R293(7.56) and I304(8.57) were found to be essential, since mutation of these amino acid residues to alanine led to a complete loss of function. Western blot analysis showed that mutant receptor R293(7.56)A was not expressed, whereas the other proteins were. Amino acid residues that are also important in receptor activation are: N286(7.49), V289(7.52), Y292(7.55), N294(8.47), F297(8.50), R298(8.51), H302(8.55) and R307(8.60). The mutation Y290(7.53)F lost 50% of efficacy, while F297(8.50)A behaved similar to wild type receptor. The double mutation, Y290(7.53)F/F297(8.50)Y, lost around 70% of efficacy and displayed a lower potency for the reference agonist 5'-(N-ethylcarboxamido)adenosine (NECA). This study provides new insight into the molecular interplay and impact of TM7 and helix 8 for hA2B receptor activation, which may be extrapolated to other adenosine receptors and possibly to other GPCRs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Activation of the adenosine A2B receptor (A2BR) can reduce myocardial ischemia/reperfusion (IR) injury. However, the mechanism underlying the A2BR-mediated cardioprotection is less clear. The present study was designed to investigate the potential mechanisms of cardioprotection mediated by A2BR., Methods and Results: C57BL/6 mice underwent 40-minute ischemia and 60-minute reperfusion. ATL-801, a potent selective A2BR antagonist, could not block ischemic preconditioning induced protection. BAY 60-6583, a highly selective A2BR agonist, significantly reduced myocardial infarct size, and its protective effect could be blocked by either ATL-801 or wortmannin. BAY 60-6583 increased phosphorylated Akt (p-Akt) levels in the heart at 10 min of reperfusion, and this phosphorylation could also be blocked by ATL-801 or wortmannin. Furthermore, BAY 60-6583 significantly increased M2 macrophages and decreased M1 macrophage and neutrophils infiltration in reperfused hearts, which also could be blocked by wortmannin. Meanwhile, confocal imaging studies showed that the majority of Akt phosphorylation in the heart was colocalized to CD206+ cells in both control and BAY 60-6583 pretreated hearts., Conclusion: Our results indicated that pretreatment with BAY 60-6583 protects the heart against myocardial IR injury by its anti-inflammatory effects, probably by modulating macrophages phenotype switching via a PI3K/Akt pathway.

G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Most, if not all, GPCRs also display a basal level of biological response in the absence of such a stimulus. This level of so-called constitutive activity results from a delicate energy equilibrium that exists between the active and the inactive state of the receptor and is the first determinant in the GPCR activation mechanism. Here we describe new insights in specific regions of the adenosine A2B receptor that are essential in activation and inactivation. We developed a new screening method using the MMY24 S. Cerevisiae strain by which we were able to screen for constitutively inactive mutants receptors (CIMs). We applied this screening method on a mutagenic library of the adenosine A2B receptor, where random mutations were introduced in transmembrane domains four and five (TM4 and TM5) linked by extracellular loop 2 (EL2). The screen resulted in the identification of 22 single and double mutant receptors, all showing a decrease in constitutive activity as well as in agonist potency. By comparing these results with a previous screen of the same mutagenic library for constitutively active mutant receptors (CAMs), we discovered specific regions in this G protein-coupled receptor involved in either inactivation or activation or both. The results suggest the activation mechanism of GPCRs to be much less restricted to sites of high conservation or direct interaction with the ligand or G protein and illustrate how dynamic the activation process of GPCRs is., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα 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 A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα 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 hA2BR, 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.

G protein-coupled A(2B) adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A(2B)AR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A(2B)AR. In cAMP accumulation assays, both 5'-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5'-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A(2B)AR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A(2B)AR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N⁶-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A(2B)AR antagonist in MIN-6 mouse pancreatic β cells expressing low A(2B)AR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target., (Published by Elsevier Inc.)

A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

Adenosine A(2B) receptors, which play a role in inflammation and cancer, are of considerable interest as novel drug targets. To gain deeper insights into ligand binding and receptor activation, we exchanged amino acids predicted to be close to the binding pocket. The alanine mutants were stably expressed in CHO cells and characterized by radioligand binding and cAMP assays using three structural classes of ligands: xanthine (antagonist), adenosine, and aminopyridine derivatives (agonists). Asn282(7.45) and His280(7.43) were found to stabilize the binding site by intramolecular hydrogen bond formation as in the related A(2A) receptor subtype. Trp247(6.48), Val250(6.51), and particularly Ser279(7.42) were shown to be important for binding of nucleosidic agonists. Leu81(3.28), Asn186(5.42), and Val250(6.51) were discovered to be crucial for binding of the xanthine-derived antagonist PSB-603. Leu81(3.28), which is not conserved among adenosine receptor subtypes, may be important for the high selectivity of PSB-603. The N186(5.42)A mutant resulted in an increased potency for agonists. The interactions of the non-nucleosidic agonist BAY60-6583 were different from those of the nucleosides: while BAY60-6583 appeared not to interact with Ser279(7.42), its interactions with Trp247(6.48) and Val250(6.51) were significantly weaker compared to those of NECA. Moreover, our results discount the hypothesis of Trp247(6.48) serving as a "toogle switch" because BAY60-6583 was able to activate the corresponding mutant. This study reveals distinct interactions of structurally diverse ligands with the human A(2B) receptor and differences between closely related receptor subtypes (A(2B) and A(2A)). It will contribute to the understanding of G protein-coupled receptor function and advance A(2B) receptor ligand design.

Objectives: Adenosine is known to induce a bronchospasm in asthma- and COPD patients. The role of A(2B) receptors was investigated with respect to several parameters of the respiratory tract: tonus of smooth muscle, ciliary beat frequency as measured by high-speed video camera connected to a microscope (both in rats) and mucociliary clearance (MCC; transport of a fluorescent dye using a microdialysis procedure) in mice., Key Findings:  NECA (5'-N-ethylcarboxamidoadenosine) (a non-selective adenosine receptor agonist) was able to acutely induce a contraction, which was reversed to a relaxation after repeated dosing. This relaxation was completely abolished by PSB-1115, an A(2B) receptor antagonist. IL-13 (cytokine) was not involved mediating acute contractility effects. MCC was increased by BAY 60-6583 (A(2B) receptor agonist) and NECA (counteracted by the A(2B) receptor antagonist PSB-1115). Activation of A(2B) adenosine receptors by BAY 60-6583 induced an increase of the ciliary beat frequency, which could be reduced by administration of PSB-1115. Several cytokines were increased by NECA although only some are relevant because they are not blocked by A(2B) receptor antagonism., Conclusions: The A(2B) receptors are involved in airway relaxation, MCC improvement and ciliary beat frequency. A(2B) receptor agonists may be of therapeutic value and should be developed., (© 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.)

Adenosine is a ubiquitous neuromodulator for which four subtypes are known (A1, A2a, A2b, and A3). Adenosine receptors (ARs) are members of the superfamily of G protein-coupled receptors. Knowledge of the 3D structure of ARs is a fundamental tool for studying how they work and for designing newer, more potent, and selective ligands. Here, we describe the procedures for modeling ARs. The starting point is represented by the resolved X-ray crystallographic structures of the A2a adenosine receptor (AA2AR) while, in the case of the other subtypes (A1, A2b, and A3), homology modeling procedures using AA2AR as a template are necessary. Minimizations and MD simulations are the computational methods used for structural refinement. The ligand-receptor interaction is studied through molecular docking simulations. The necessity of validating the obtained computational models through all of the available experimental data is outlined. Finally, the emerging importance of the homo- and heterodimerization of ARs is taken into account and a procedure for modeling a dimer is reported., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The nuclear factor kappa B (NFκB) pathway controls a variety of processes, including inflammation, and thus, the regulation of NFκB has been a continued focus of study. Here, we report a newly identified regulation of this pathway, involving direct binding of the transcription factor NFκB1 (the p105 subunit of NFκB) to the C-terminus of the A(2B) adenosine receptor (A(2B)AR), independent of ligand activation. Intriguingly, binding of A(2B)AR to specific sites on p105 prevents polyubiquitylation and degradation of p105 protein. Ectopic expression of the A(2B)AR increases p105 levels and inhibits NFκB activation, whereas p105 protein levels are reduced in cells from A(2B)AR-knockout mice. In accordance with the known regulation of expression of anti- and pro-inflammatory cytokines by p105, A(2B)AR-null mice generate less interleukin (IL)-10, and more IL-12 and tumor necrosis factor (TNF-α). Taken together, our results show that the A(2B)AR inhibits NFκB activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation. Our findings unveil a surprising function for the A(2B)AR, and provide a novel mechanistic insight into the control of the NFκB pathway and inflammation.

The four subtypes of adenosine receptors form relevant drug targets in the treatment of, e.g., diabetes and Parkinson's disease. In the present study, we aimed at finding novel small molecule ligands for these receptors using virtual screening approaches based on proteochemometric (PCM) modeling. We combined bioactivity data from all human and rat receptors in order to widen available chemical space. After training and validating a proteochemometric model on this combined data set (Q(2) of 0.73, RMSE of 0.61), we virtually screened a vendor database of 100910 compounds. Of 54 compounds purchased, six novel high affinity adenosine receptor ligands were confirmed experimentally, one of which displayed an affinity of 7 nM on the human adenosine A(1) receptor. We conclude that the combination of rat and human data performs better than human data only. Furthermore, we conclude that proteochemometric modeling is an efficient method to quickly screen for novel bioactive compounds.

The adenosine A(2B) receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A(2B) receptor with dithiothreitol led to a 74-fold increase in the EC(50) value of the agonist NECA in cyclic AMP accumulation. In the C78(3.25)S and the C171(45.50)S mutant high-affinity binding of the A(2B) antagonist radioligand [(3)H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25-C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A(2B) receptors. In contrast, the C166(45.45)S and the C167(45.46)S mutant as well as the C166(45.45)S-C167(45.46)S double mutant behaved like the wild-type receptor, while in the C154(45.33)S mutant significant, although more subtle effects on cAMP accumulation were observed - decrease (BAY60-6583) or increase (NECA) - depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A(2A) receptor, which showed four disulfide bonds, the present data indicate that in the A(2B) receptor only the C3.25-C45.50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine residues in the ECL2 of the A(2B) receptor not involved in stabilization of the receptor structure may have other functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The crystal structure of the human A(2A) adenosine receptor, a member of the G protein-coupled receptor (GPCR) family, is used as a starting point for the structural characterization of the conformational equilibrium around the inactive conformation of the human A(2) (A(2A) and A(2B)) adenosine receptors (ARs). A homology model of the closely related A(2B)AR is reported, and the two receptors were simulated in their apo form through all-atom molecular dynamics (MD) simulations. Different conditions were additionally explored in the A(2A)AR, including the protonation state of crucial histidines or the presence of the cocrystallized ligand. Our simulations reveal the role of several conserved residues in the ARs in the conformational equilibrium of the receptors. The "ionic lock" absent in the crystal structure of the inactive A(2A)AR is rapidly formed in the two simulated receptors, and a complex network of interacting residues is presented that further stabilizes this structural element. Notably, the observed rotameric transition of Trp6.48 ("toggle switch"), which is thought to initiate the activation process in GPCRs, is accompanied by a concerted rotation of the conserved residue of the A(2)ARs, His6.52. This new conformation is further stabilized in the two receptors under study by a novel interaction network involving residues in transmembrane (TM) helices TM5 (Asn5.42) and TM3 (Gln3.37), which resemble the conformational changes recently observed in the agonist-bound structure of β-adrenoreceptors. Finally, the interaction between Glu1.39 and His7.43, a pair of conserved residues in the family of ARs, is found to be weaker than previously thought, and the role of this interaction in the structure and dynamics of the receptor is thoroughly examined. All these findings suggest that, despite the commonalities with other GPCRs, the conformational equilibrium of ARs is also modulated by specific residues of the family.

The highly variable extracellular loops in G protein-coupled receptors (GPCRs) have been implicated in receptor activation, the mechanism of which is poorly understood. In a random mutagenesis screen on the human adenosine A(2B) receptor (A(2B)R) using the MMY24 Saccharomyces cerevisiae strain as a read-out system, we found that two residues in the first extracellular loop, a phenylalanine and an aspartic acid at positions 71 and 74, respectively, are involved in receptor activation. We subsequently performed further site-directed and site-saturation mutagenesis. These experiments revealed that the introduction of mutations at either of the identified positions results in a wide variety of receptor activation profiles, with changes in agonist potency, constitutive activity, and intrinsic activity. Radioligand binding studies showed that the changes in activation were not due to changes in receptor expression. We interpret these data in the light of the recently revealed structure of the adenosine A(2A)R, the closest homologue of the A(2B)R. The two residues are suggested to be vital in maintaining the tertiary structure of a β sheet in the extracellular domain of the A(2B)R. We hypothesize that deterioration of structure in the extracellular domains of GPCRs compromises overall receptor structure with profound consequences for receptor activation and constitutive activity.

This research was carried out to study the pharmacological activity of a newly synthesized series of 2-alkoxy-[1,2,4]triazolo[1,5-a]quinazolin-5-ones as adenosine receptor antagonists. These compounds have been tested in radioligand binding assays on cloned Chinese hamster ovary (CHO) cells transfected with A(1), A(2A), A(2B) and A(3) receptors. In particular, among the triazoloquinazolines (1-11), the dialkoxy derivative (7b) was found to have the highest affinity at A(1) subtype receptor, and its radioligand binding activity together with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was studied. Finally, the structure-activity relationship (SAR) studies on the titled compounds provide some new insights about steric hindrance and lipophilic requirements for anchoring to the adenosine receptors recognition site.

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM)., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A series of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones was synthesized and tested in radioligand binding assays to determine their affinities for the human adenosine A(1), A(2A), A(2B) and A(3) receptors. Results indicated that this scaffold is appropriate for adenosine receptor subtype A(1) ligands and that the best arranged groups around this scaffold are 3- and 4-pyridinyl at position 1, benzyl at position 3, hydrogen at position 6 and 3-thienyl or phenyl at position 9. The most interesting compounds showed K(i) for A1 in the nanomolar range and an appreciable selectivity for other receptor subtypes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ligand-based and receptor-based methods were used to investigate the binding modes of human adenosine A(2B) antagonists. At first, pharmacophore models were developed based on 140 diverse A(2B) antagonists from literature. Meanwhile, the structural model of A(2B) receptor was built up based on the crystal structure of human A(2A) receptor and validated by Induced Fit docking, Glide-XP and Glide-SP docking. Two models matched each other very well and some important implications were hence obtained. The residues of Phe173 and Glu174 in the second extracellular loop and Asn254 were crucial to the antagonists binding to form pi-pi stacking and hydrogen-bonding interactions. These findings would be very helpful for the discovery of novel and potent A(2B) antagonists., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

One of the grand challenges in chemical biology is identifying a small-molecule modulator for each individual function of all human proteins. Instead of targeting one protein at a time, an efficient approach to address this challenge is to target entire protein families by taking advantage of the relatively high levels of chemical promiscuity observed within certain boundaries of sequence phylogeny. We recently developed a computational approach to identifying the potential protein targets of compounds based on their similarity to known bioactive molecules for almost 700 targets. Here, we describe the direct identification of novel antagonists for all four adenosine receptor subtypes by applying our virtual profiling approach to a unique synthesis-driven chemical collection composed of 482 biologically-orphan molecules. These results illustrate the potential role of in silico target profiling to guide efficiently screening campaigns directed to discover new chemical probes for all members of a protein family., ((c) 2010 Elsevier Ltd. All rights reserved.)

A(2B) adenosine receptors have been investigated in recent years as potential target for the treatment of different pathologies. The involvement of this receptor in processes such as interleukins secretion, Ca(2+) mobilization, hepatic glucose regulation, tumor vascularisation, and cardioprotection have stimulated many researchers to develop specific agonists and antagonists. For many years, the lack of potent and selective A(2B) ligands precluded a deep exploration of their therapeutic prospective; at present, much progress in the field of antagonists led to preclinical studies for different compounds. Less populated is the universe of A(2B) agonists, but really promising for the involvement in ischemic preconditioning. A summary of the most significant advancements in the synthesis of new compounds and of the principal structure activity relationships is reported. The xanthine-based A(2B) antagonists currently show the better profile of affinity and selectivity, as CVT-6883 (CVT-Therapeutics: K(i(A2B))=22 nM, and selectivity higher than 50-fold over other subtypes), MRE-2029-F20 (Baraldi's group: K(i(A2B))=5.5 nM, selectivity >180 fold), LAS38096 (Almirall Prodesfarma: K(i(A2B))=17 nM, selectivity >60 fold), OSIP339391 (OSI Pharmaceuticals: K(i(A2B))=0.5 nM, selectivity >80 fold), PSB601 (Bonn University: K(i(A2B))=3.6 nM, selectivity >140 fold) and the deazaxanthine 32 of Carotti's group (K(i(A2B))=11 nM, selectivity >90 fold). Other recently emerging scaffolds with promising biological profiles are described. With regard to the agonists, many research groups are involved in the discovery of useful agonist radioligands, but the only example of potent and rather selective A(2B) agonists are compound 65, recently synthesized, and BAY-60-6583, that is under preclinical phase investigation.

A three-dimensional model of the human adenosine A2B receptor was generated by means of homology modelling, using the crystal structures of bovine rhodopsin, the beta2-adrenergic receptor, and the human adenosine A2A receptor as templates. In order to compare the three resulting models, the binding modes of the adenosine A2B receptor antagonists theophylline, ZM241385, MRS1706, and PSB601 were investigated. The A2A-based model was much better able to stabilize the ligands in the binding site than the other models reflecting the high degree of similarity between A2A and A2B receptors: while the A2B receptor shares about 21% of the residues with rhodopsin, and 31% with the beta2-adrenergic receptor, it is 56% identical to the adenosine A2A receptor. The A2A-based model was used for further studies. The model included the transmembrane domains, the extracellular and the intracellular hydrophilic loops as well as the terminal domains. In order to validate the usefulness of this model, a docking analysis of several selective and nonselective agonists and antagonists was carried out including a study of binding affinities and selectivities of these ligands with respect to the adenosine A2A and A2B receptors. A common binding site is proposed for antagonists and agonists based on homology modelling combined with site-directed mutagenesis and a comparison between experimental and calculated affinity data. The new, validated A2B receptor model may serve as a basis for developing more potent and selective drugs.

Adenosine receptor A2B (ADoR A2B) is an important G protein-coupled receptor (GPCR) of the rhodopsin family, and plays a pivotal role in gastrointestinal, neurological and hypersensitive disorders. QSAR and pharmacophore studies were carried out using 63 ADoR A2B inhibitor molecules to characterize molecular features and structural requirements for biological interaction. QSAR modelling using genetic algorithm- partial least squares (G/PLS) method identified molecular shape, size electrophilicity and conformational flexibility as important descriptors for these compounds affinity to the receptor. Further analysis of pharmacophore model revealed hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), hydrophobic aliphatic (HY-ala) and hydrophobic aromatic (HY-aro) as the crucial molecular features that predict binding affinity of these compounds to ADoR A2B. These observations provide important insights to the rationale development of novel and potent compounds against ADoR A2B.

The binding modes at the A 2B adenosine receptor (AR) of 72 derivatives of adenosine and its 5'- N-methyluronamide with diverse substitutions at the 2 and N (6) positions were studied using a molecular modeling approach. The compounds in their receptor-docked conformations were used to build CoMFA and CoMSIA quantitative structure-activity relationship models. Various parameters, including different types of atomic charges, were examined. The best statistical parameters were obtained with a joint CoMFA and CoMSIA model: R (2) = 0.960, Q (2) = 0.676, SEE = 0.175, F = 158, and R (2) test = 0.782 for an independent test set containing 18 compounds. On the basis of the modeling results, four novel adenosine analogues, having elongated or bulky substitutions at N (6) position and/or 2 position, were synthesized and evaluated biologically. All of the proposed compounds were potent, full agonists at the A 2B AR in adenylate cyclase studies. Thus, in support of the modeling, bulky substitutions at both positions did not prevent A 2B AR activation, which predicts separate regions for docking of these moieties.

A series of [1,2,4]triazolo[1,5-c]quinazolines were prepared in satisfactory yields by reaction of some derivatives of 2-aminobenzohydrazide with several hydrochlorides of aromatic amidines, and their binding affinities for the recombinant human adenosine A2A and A2B receptors were determined. None of the new compounds showed noteworthy affinity for these receptors, though a very high affinity for the A2A receptor and, consequently, a high level of A2A/A2B selectivity was revealed for one of the synthesized compounds.