Novel curcumin analog (cis-trans curcumin) as ligand to adenosine receptors A2A and A2B: potential for therapeutics.

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 Gs-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 A2A and AR A2B with Ki values of 5 µM and 7 µM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A2B and A2A. 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 Gs-ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives.

Extracellular vesicles derived from CD73 modified human umbilical cord mesenchymal stem cells ameliorate inflammation after spinal cord injury.

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 A2bR 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-1ß, 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.

Amyloid beta (1-42) downregulates adenosine-2b receptors in addition to mitochondrial impairment and cholinergic dysfunction in memory-sensitive mouse brain regions.

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 Aß on the level of expression of A2b receptor was investigated in discrete memory-sensitive mouse brain regions. Aß (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. Aß significantly attenuated the spatial working memory in MWM and Y-maze tests. In addition, Aß 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, Aß 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.

Exploiting the Indole Scaffold to Design Compounds Binding to Different Pharmacological Targets.

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 (GABAA) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A2B adenosine receptor (A2B 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.

Stimulation of the A2B Adenosine Receptor Subtype Enhances Connexin26 Hemichannel Activity in Small Airway Epithelial Cells.

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 La3+, 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 A2B 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 A2B 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.

Protective Effects of Oxymatrine on Vascular Endothelial Cells from High-Glucose-Induced Cytotoxicity by Inhibiting the Expression of A2B Receptor.

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-1ß 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-1ß 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.

Tritium-labeled agonists as tools for studying adenosine A2B receptors.

A selective agonist radioligand for A2B adenosine receptors (A2BARs) 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 A2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [3H]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 A2BAR agonist [3H]NECA for its potential to label A2BARs. [3H]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 A2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [3H]NECA than versus the A2B-antagonist radioligand [3H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A2BAR agonist, it displayed higher affinity versus [3H]PSB-603 than versus [3H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A2BAR. In conclusion, [3H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A2BAR conformation.

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

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N1 (1,2) or N10 (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A1 and A2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A1 and A2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A1 AR and A2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A1 and A2B 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.

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor.

On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A 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 A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 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 [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]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.

Actinin-1 binds to the C-terminus of A2B adenosine receptor (A2BAR) and enhances A2BAR cell-surface expression.

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.

A novel mechanism of control of NFκB activation and inflammation involving A2B adenosine receptors.

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.

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

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian 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.

Structural insight into the dual-antagonistic mechanism of AB928 on adenosine A2 receptors.

The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR 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 A2AR complexed with AB928 and a selective A2AR antagonist 2-118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2-118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study.

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 A2B receptor (A2BAR). 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 A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, 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.

Ligand-specific binding and activation of the human adenosine A(2B) receptor.

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.

Classifier ensemble based on feature selection and diversity measures for predicting the affinity of A(2B) adenosine receptor antagonists.

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.

GPCR structure and activation: an essential role for the first extracellular loop in activating the adenosine A2B receptor.

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 ß 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.

In silico development of adenosine A2B receptor antagonists for sickle cell disease.

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 ß 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 (O2), 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.

A patent review of adenosine A2B receptor antagonists (2016-present).

INTRODUCTION: A2B adenosine receptor (A2BAR) 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 A2A and A2BARs 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 A2BAR or A2A/A2BARs. 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 A2B or A2A/A2B receptor ligands is reported. EXPERT OPINION: Among the four P1 receptors, A2BAR 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 A2BAR and A2A/A2BARs drugs. Although several compounds have been synthesized only a few molecules have entered clinical trials.

Molecular dynamics simulations reveal insights into key structural elements of adenosine receptors.

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 ß-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.