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Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A 1 R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A 1 R of an A 1 R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A 1 R antagonists and showed evidence of covalent binding and simultaneous binding across an A 1 R homodimer. Greater selectivity for A 1 R over the adenosine A 3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A 1 AR in living cells. We pharmacologically demonstrate covalent labeling of A 1 AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A 1 AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A 1 ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A 1 AR in more physiologically relevant environments.

Background/aim: Recent research suggests that adenosine receptors (ARs) influence many of the metabolic abnormalities associated with diabetes. A non-xanthine benzylidene indanone derivative 2-(3,4-dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1 H-inden-1-one (2-BI), has shown to exhibit higher affinity at A 1 /A 2A ARs compared to caffeine. Due to its structural similarity to caffeine, and the established antidiabetic effects of caffeine, the current study was initiated to explore the possible antidiabetic effect of 2-BI., Methods: The study was designed to assess the antidiabetic effects of several A 1 and/or A 2A AR antagonists, via intestinal glucose absorption and glucose-lowering effects in fructose-streptozotocin (STZ) induced diabetic rats. Six-week-old male Sprague-Dawley rats were induced with diabetes via fructose and streptozotocin. Rats were treated for 4 weeks with AR antagonists, metformin and pioglitazone, respectively. Non-fasting blood glucose (NFBG) was determined weekly and the oral glucose tolerance test (OGTT) was conducted at the end of the intervention period., Results: Dual A 1 /A 2A AR antagonists (caffeine and 2-BI) decreased glucose absorption in the intestinal membrane significantly (p < 0.01), while the selective A 2A AR antagonist (Istradefylline), showed the highest significant (p < 0.001) reduction in intestinal glucose absorption. The selective A 1 antagonist (DPCPX) had the least significant (p < 0.05) reduction in glucose absorption. Similarly, dual A 1 /A 2A AR antagonists and selective A 2A AR antagonists significantly reduced non-fast blood glucose and improved glucose tolerance in diabetic rats from the first week of the treatment. Conversely, the selective A 1 AR antagonist did not reduce non-fast blood glucose significantly until the 4th week of treatment. 2-BI, caffeine and istradefylline compared well with standard antidiabetic treatments, metformin and pioglitazone, and in some cases performed even better., Conclusion: 2-BI exhibited good antidiabetic activity by reducing intestinal postprandial glucose absorption and improving glucose tolerance in a diabetic animal model. The dual antagonism of A 1 /A 2A ARs presents a positive synergism that could provide a new possibility for the treatment of diabetes., (© 2023. The Author(s).)

Over the past few years, great progress has been made in the development of high-affinity adenosine A 1 and/or A 2A receptor antagonists-promising agents for the potential treatment of Parkinson's disease. Unfortunately, many of these compounds raise structure-related concerns. The present study investigated the effect of ring closures on the rA 1 /A 2A affinity of compounds containing a highly reactive α,β-unsaturated carbonyl system, hence providing insight into the potential of heterocycles to address these concerns. A total of 12 heterocyclic compounds were synthesised and evaluated in silico and in vitro. The test compounds performed well upon qualitative assessment of drug-likeness and were generally found to be free from potentially problematic fragments. Most also showed low/weak cytotoxicity. Results from radioligand binding experiments confirm that heterocycles (particularly 2-substituted 3-cyanopyridines) can replace the promiscuous α,β-unsaturated ketone functional group without compromising A 1 /A 2A affinity. Structure-activity relationships highlighted the importance of hydrogen bonds in binding to the receptors of interest. Compounds 3c (rA 1 K i  = 16 nM; rA 2A K i  = 65 nM) and 8a (rA 1 K i  = 102 nM; rA 2A K i  = 37 nM), which both act as A 1 antagonists, showed significant dual A 1 /A 2A affinity and may, therefore, inspire further investigation into heterocycles as potentially safe and potent adenosine receptor antagonists., (© 2021 John Wiley & Sons Ltd.)

We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A 1 AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarilly evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure-activity and structure-selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R 4 and R 6 of the pyrimidine core but most importantly the prominent role to the unprecedented A 1 AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure-activity relationship trends on both A 1 and A 2A ARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1,2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A 1 receptor (A 1 R) and adenosine A 3 receptor (A 3 R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A 1 R and A 3 R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A 2A and A 2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A 1 R and A 3 R localization and trafficking in native cells and living systems.

The adenosine A 1 receptor (A 1 AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A 1 AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A 1 AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A 1 AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A 1 AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A 1 AR molecular pharmacology and signaling in living cells.

Adenosine acts as an endogenous anticonvulsant and seizure terminator in the brain. Many of its anticonvulsive effects are mediated through the activation of the adenosine A 1 receptor, a G protein-coupled receptor with a wide array of targets. Activating A 1 receptors is an effective approach to suppress seizures. This review gives an overview of the neuronal targets of the adenosine A 1 receptor focusing in particular on signaling pathways resulting in neuronal inhibition. These include direct interactions of G protein subunits, the adenyl cyclase pathway and the phospholipase C pathway, which all mediate neuronal hyperpolarization and suppression of synaptic transmission. Additionally, the contribution of the guanyl cyclase and mitogen-activated protein kinase cascades to the seizure-suppressing effects of A 1 receptor activation are discussed. This review ends with the cautionary note that chronic activation of the A 1 receptor might have detrimental effects, which will need to be avoided when pursuing A 1 receptor-based epilepsy therapies.

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A 1 receptor (hA 1 AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA 1 AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA 1 AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study. In a radioligand binding assay, LUF7746's apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA 1 AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA 1 AR demonstrated that amino acid Y271 7.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746's irreversible activation of an A 1 receptor-mediated cell morphological response. These results led us to conclude that LUF7746 is a novel covalent hA 1 AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin-chalcone hybrids were synthesized (compounds 1 - 8 ) and screened in radioligand binding ( h A 1 , h A 2A and h A 3 ) and adenylyl cyclase ( h A 2B ) assays in order to evaluate their affinity for the four human AR subtypes ( h ARs). Coumarin-chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for h A 1 or h A 3 subtypes. In general, hydroxy-substituted hybrids showed some affinity for the h A 1 , while the methoxy counterparts were selective for the h A 3 . The most potent h A 1 ligand was compound 7 ( K i = 17.7 µM), whereas compound 4 was the most potent ligand for h A 3 ( K i = 2.49 µM). In addition, docking studies with h A 1 and h A 3 homology models were established to analyze the structure-function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D 1 - and adenosine A 1 -receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A 1 -receptor (A 1 R), A 1 R-G279S 7.44 , was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A 1 -receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A 1 R-G279S 7.44 was enhanced by about 2 and 8 K when compared with wild-type A 1 -receptor and A 1 R-Y288A 7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A 1 R-G279S 7.44 (73 ± 23 kJ/mol) than in wild-type A 1 R (135 ± 4 kJ/mol) or in A 1 R-Y288A 7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A 1 R-G279S 7.44 was more effective in promoting guanine nucleotide exchange than wild-type A 1 R. We detected similar levels of complexes formed between D 1 -receptors and wild-type A 1 R or A 1 R-G279S 7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D 1 -receptor and A 1 R-G279S 7.44 than in those coexpressing wild-type A 1 R. These observations predict enhanced inhibition of dopaminergic signaling by A 1 R-G279S 7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A 1 -receptor antagonizes responses elicited by dopamine D 1 -receptor. We show that this activity is more pronounced in a mutant version of the A 1 -receptor (A 1 R-G279S 7.44 ), which was identified in individuals suffering from early-onset Parkinson's disease., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 by The Author(s).)

Among still comparatively few G protein-coupled receptors, the adenosine A 2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A 1 receptor (A 1 AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A 2A AR and the A 3 AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A 3 AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Adenosine receptors (ARs), like many otherGprotein-coupledreceptors (GPCRs), are targets of primary interest indrug design. However, one of the main limits for the development of drugs for this class of GPCRs is the complex selectivity profile usually displayed by ligands. Numerous efforts have been madefor clarifying the selectivity of ARs, leading to the development of many ligand-based models. The structure of the AR subtype A 1 (A 1 AR) has been recently solved,providing important structural insights. In the present work, we rationalized the selectivity profile of two selective A 1 AR and A 2A AR antagonists, investigating their recognition trajectories obtained by Supervised Molecular Dynamics from an unbound state and monitoring the role of the water molecules in the binding site., Competing Interests: The authors declare no conflict of interest.

The receptorial responsiveness method (RRM) is a procedure that is based on a simple nonlinear regression while using a model with two variables (X, Y) and (at least) one parameter to be determined (c x ). The model of RRM describes the co-action of two agonists that consume the same response capacity (due to the use of the same postreceptorial signaling in a biological system). While using RRM, uniquely, an acute increase in the concentration of an agonist (near the receptors) can be quantified (as c x ), via evaluating E/c curves that were constructed with the same or another agonist in the same system. As this measurement is sensitive to the implementation of the curve fitting, the goal of the present study was to test RRM by combining different ways and setting options, namely: individual vs. global fitting, ordinary vs. robust fitting, and three weighting options (no weighting vs. weighting by 1/Y 2 vs. weighting by 1/SD 2 ). During the testing, RRM was used to estimate the known concentrations of stable synthetic A 1 adenosine receptor agonists in isolated, paced guinea pig left atria. The estimates were then compared to the known agonist concentrations (to assess the accuracy of RRM); furthermore, the 95% confidence limits of the best-fit values were also considered (to evaluate the precision of RRM). It was found that, although the global fitting offered the most convenient way to perform RRM, the best estimates were provided by the individual fitting without any weighting, almost irrespective of the fact whether ordinary or robust fitting was chosen.

The adenosine A1 receptor (A1R) is one of four adenosine receptors in humans, which are involved in the function of the cardiovascular, respiratory and central nervous systems. Experimental results indicate that A1R can form a homodimer and that the protomer-protomer interaction in the A1R dimer is related to certain pharmacological characteristics of A1R activation. In this work, we performed docking, metadynamics simulation, conventional molecular dynamics simulations, Gaussian-accelerated molecular dynamics simulations, potential of mean force calculations, dynamic cross-correlation motions analysis and community network analysis to study the binding mode of 5'-N-ethylcarboxamidoadenosine (NECA) to A1R and the effect of dimerization on the activation of A1R. Our results show that NECA binds to A1R in a similar mode to adenosine in the A1R crystal structure and NECA in the A2AR crystal structure. The A1R homodimer can be activated by one or two agonists with NECA occupying its orthosteric pockets in one (which we call the NECA-A1R system) or both protomers (which we call the dNECA-A1R system). In the NECA-A1R system, activation is predicated in the protomer without NECA bound. In the dNECA-A1R system, only one protomer achieves the active state. These findings suggest an asymmetrical activation mechanism of the homodimer and a negative cooperativity between the two protomers. We envision that our results may further facilitate the drug development of A1R.

Previous studies explored 2-benzylidine-1-tetralone derivatives as innovative adenosine A 1 and A 2A receptor antagonists for alternative non-dopaminergic treatment of Parkinson's disease. This study's aim is to investigate structurally related 2-benzylidene-1-indanones with substitutions on ring A and B as novel, potent and selective adenosine A 1 and A 2A receptor blockers. 2-Benzylidene-1-indanone derivatives were synthesised via acid catalysed aldol condensation reactions and evaluated via radioligand binding assays to ascertain structure activity relationships to govern A 1 and A 2A AR affinity. The results indicated that hydroxy substitution at C4 of ring A and meta (3'), or para (4') substitution on ring B of the 2-benzylidene-1-indanone scaffold (2C: ) is preferred over substitution at C5 (2D: ) or C6 (2E: ) of ring A for adenosine A 1 receptor activity and selectivity in the micromolar range. Furthermore, substitution at the meta (3') position of ring B with chlorine lead to the highly potent and selective adenosine A 2A receptor antagonist, compound 2 H: . Compound 2C: and the 2Q: behaved as adenosine A 1 receptor antagonists in the performed GTP shift assays. In view of these findings, compounds 2C: , 2 H: , 2Q: and 2P: are potent and selective adenosine A 1 and A 2A receptor antagonists for the potential treatment of neurological conditions., Competing Interests: The authors have no conflict of interest to declare., (© Georg Thieme Verlag KG Stuttgart · New York.)

In earlier studies, we generated concentration-response (E/c) curves with CPA ( N 6 -cyclopentyladenosine; a selective A 1 adenosine receptor agonist) or adenosine, in the presence or absence of S -(2-hydroxy-5-nitrobenzyl)-6-thioinosine (NBTI, a selective nucleoside transport inhibitor), and with or without a pretreatment with 8-cyclopentyl- N 3 -[3-(4-(fluorosulfonyl)-benzoyloxy)propyl]- N 1 -propylxanthine (FSCPX, a chemical known as a selective, irreversible A 1 adenosine receptor antagonist), in isolated, paced guinea pig left atria. Meanwhile, we observed a paradoxical phenomenon, i.e. the co-treatment with FSCPX and NBTI appeared to enhance the direct negative inotropic response to adenosine. In the present in silico study, we aimed to reproduce eight of these E/c curves. Four models (and two additional variants of the last model) were constructed, each one representing a set of assumptions, in order to find the model exhibiting the best fit to the ex vivo data, and to gain insight into the paradoxical phenomenon in question. We have obtained in silico evidence for an interference between effects of FSCPX and NBTI upon our ex vivo experimental setting. Regarding the mechanism of this interference, in silico evidence has been gained for the assumption that FSCPX inhibits the effect of NBTI on the level of endogenous (but not exogenous) adenosine. As an explanation, it may be hypothesized that FSCPX inhibits an enzyme participating in the interstitial adenosine formation. In addition, our results suggest that NBTI does not stop the inward adenosine flux in the guinea pig atrium completely., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A 1 -receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A 1 -receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A 1 -receptor agonists., Experimental Approach: A Nano-luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N-terminal Nluc-tagged A 1 -receptors expressed in HEK293T cells in conjunction with both fluorescent A 1 -receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645., Key Results: PD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A 1 -receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A 1 -receptor agonists in a species-dependent manner that involved increases in B max and pK D ., Conclusions and Implications: These results demonstrate the power of the NanoBRET ligand-binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A 1 -receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A 1 -receptors to an active agonist conformation (R*)., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Dopaminergic and purinergic signaling play a pivotal role in neurological diseases associated with motor symptoms, including Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, Huntington disease, Restless Legs Syndrome (RLS), spinal cord injury (SCI), and ataxias. Extracellular dopamine and adenosine exert their functions interacting with specific dopamine (DR) or adenosine (AR) receptors, respectively, expressed on the surface of target cells. These receptors are members of the family A of G protein-coupled receptors (GPCRs), which is the largest protein superfamily in mammalian genomes. GPCRs are target of about 40% of all current marketed drugs, highlighting their importance in clinical medicine. The striatum receives the densest dopamine innervations and contains the highest density of dopamine receptors. The modulatory role of adenosine on dopaminergic transmission depends largely on the existence of antagonistic interactions mediated by specific subtypes of DRs and ARs, the so-called A 2A R-D 2 R and A 1 R-D 1 R interactions. Due to the dopamine/adenosine antagonism in the CNS, it was proposed that ARs and DRs could form heteromers in the neuronal cell surface. Therefore, adenosine can affect dopaminergic signaling through receptor-receptor interactions and by modulations in their shared intracellular pathways in the striatum and spinal cord. In this work we describe the allosteric modulations between GPCR protomers, focusing in those of adenosine and dopamine within the A 1 R-D 1 R heteromeric complex, which is involved in RLS. We also propose that the knowledge about the intricate allosteric interactions within the A 1 R-D 1 R heterotetramer, may facilitate the treatment of motor alterations, not only when the dopamine pathway is hyperactivated (RLS, chorea, etc.) but also when motor function is decreased (SCI, aging, PD, etc.)., (© 2019 Elsevier Inc. All rights reserved.)

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.)

Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A 1 receptor (A 1 AR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the A 1 AR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the A 1 AR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the A 1 AR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the A 1 AR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the A 1 AR, which will greatly facilitate the receptor structure-based drug design.

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A 1 , A 2A , A 2B and A 3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA 1 and hA 2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A 1 /A 2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA 1 K i  = 10.2 nM; hA 2A K i  = 4.72 nM) and behaved as a potent A 1 /A 2A antagonist/inverse agonist (hA 1 IC 50  = 13.4 nM; hA 2A IC 50  = 5.34 nM)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The class A adenosine A 1 receptor (A 1 R) is a G-protein-coupled receptor that preferentially couples to inhibitory G i/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A 1 R in complex with adenosine and heterotrimeric G i2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A 1 R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A 1 R primarily via amino acids in the C terminus of the Gα i α5-helix, concomitant with a 10.5 Å outward movement of the A 1 R transmembrane domain 6. Comparison with the agonist-bound β 2 adrenergic receptor-G s -protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A 1 R structure provides molecular insights into receptor and G-protein selectivity.

We examined whether orexin neurons might play a protective role against fasting- and adenosine-induced hypothermia. We first measured body temperature (BT) in orexin neuron-ablated (ORX-AB) mice and wild-type (WT) controls during 24 hours of fasting. As expected, the magnitude of BT drop and the length of time suffering from hypothermia were greater in ORX-AB mice than in WT mice. Orexin neurons were active just before onset of hypothermia and during the recovery period as revealed by calcium imaging in vivo using G-CaMP. We next examined adenosine-induced hypothermia via an intracerebroventricular administration of an adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA), which induced hypothermia in both ORX-AB and WT mice. The dose of CHA required to initiate a hypothermic response in ORX-AB mice was more than 10 times larger than the dose for WT mice. Once hypothermia was established, the recovery was seemingly slower in ORX-AB mice. Activation of orexin neurons during the recovery phase was confirmed by immunohistochemistry for c-Fos. We propose that orexin neurons play dual roles (enhancer in the induction phase and compensator during the recovery phase) in adenosine-induced hypothermia and a protective/compensatory role in fasting-induced hypothermia.

Our previous work discovered that combining the appropriate 5'- and N 6 -substitution in adenosine derivatives leads to the highly selective human A 1 adenosine receptor (hA 1 AR) agonists or highly potent dual hA 1 AR agonists and hA 3 AR antagonists. In order to explore novel dual adenosine receptor ligands, a series of N 6 -substituted-5'-pyrazolyl-adenosine and 2-chloro-adenosine derivatives were synthesized and assayed in vitro at all ARs. The N 6 -(±)-endo-norbornyl derivative 12 was the most potent and selective at A 1 AR and effective as an analgesic in formalin test in mice, but none of the 5'-pyrazolyl series compounds showed a dual behavior at hA 1 and hA 3 AR. Molecular modeling studies rationalized the structure-activity relationships and the selectivity profiles of the new series of A 1 AR agonists. Interestingly, an unexpected inverted binding mode of the N 6 -tetrahydrofuranyl derivative 14 was hypothesized to explain its low affinity at A 1 AR.

Antagonists of the adenosine receptors (A 1 and A 2A ) are thought to be beneficial in neurological disorders, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore 2-benzylidene-1-tetralone derivatives as antagonists of A 1 and/or A 2A adenosine receptors. In general, the test compounds were found to be selective for the A 1 adenosine receptor, with only three test compounds possessing affinity for both the A 1 and A 2A adenosine receptor. The 2-benzylidene-1-tetralones bearing a hydroxyl substituent at either position C5, C6 or C7 of ring A displayed favourable adenosine A 1 receptor binding, while C5 hydroxy substitution led to favourable A 2A adenosine receptor affinity. Interestingly, para-hydroxy substitution on ring B in combination with ring A bearing a hydroxy at position C6 or C7 provided the 2-benzylidene-1-tetralones with both A 1 and A 2A adenosine receptor affinity. Compounds 4 and 8 displayed the highest A 1 and A 2A adenosine receptor affinity with values below 7 μm. Both these compounds behaved as A 1 adenosine receptor antagonists in the performed GTP shift assays. In conclusion, the 2-benzylidene-1-tetralone derivatives can be considered as lead compounds to design a new class of dual acting adenosine A 1 /A 2A receptor antagonists that may have potential in treating both dementia and locomotor deficits in Parkinson's disease., (© 2017 John Wiley & Sons A/S.)

The adenosine A 1 and A 2A receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A 1 receptor at 3.3 Å resolution with PSB36, an A 1 -selective xanthine-based antagonist. This is compared with structures of the A 2A receptor with PSB36 (2.8 Å resolution), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A 1 R and A 2A R were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T270 7.35 in A 1 R (M270 7.35 in A 2A R) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A 1 R or A 2A R antagonists., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The adenosine A 1 receptor (A 1 -AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A 1 -AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A 2A receptor (A 2A -AR) structure. Mutational and computational analysis of A 1 -AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The adenosine A 1 G protein-coupled receptor (A 1 AR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the A 1 AR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of A 1 AR-ECL2 to explore the role of this domain on A 1 AR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in A 1 AR orthosteric ligand binding and receptor activation., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Three 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives (4-6) were investigated as potential non-nucleoside agonists at human adenosine receptors (ARs). When tested in competition binding experiments, these compounds exhibited low micromolar affinity (K i values comprised between 1.2 and 1.9 μm) for the A 1 AR and no appreciable affinity for the A 2A and A 3 ARs. Evaluation of their efficacy profiles by measurement of intracellular cAMP levels revealed that 4 and 5 behave as non-nucleoside agonists of the A 1 AR with EC 50 values of 0.47 and 0.87 μm, respectively. No clear concentration-response curves could be instead obtained for 6, probably because this compound modulates one or more additional targets, thus masking the putative effects exerted by its activation of A 1 AR. The three compounds were not able to modulate A 2B AR-mediated cAMP accumulation induced by the non-selective AR agonist NECA, thus demonstrating no affinity toward this receptor., (© 2016 John Wiley & Sons A/S.)

G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially βγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gβγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gβγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the βγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC., (Published by Elsevier Inc.)

A novel series of carbamate substituted 2-amino-4,6-diphenylpyrimidines was evaluated as potential dual adenosine A1 and A2A receptor antagonists. The majority of the synthesised compounds exhibited promising dual affinities, with A1Ki values ranging from 0.175 to 10.7 nM and A2AKi values ranging from 1.58 to 451 nM. The in vivo activity illustrated for 3-(2-amino-6-phenylpyrimidin-4-yl)phenyl morpholine-4-carboxylate (4c) is indicative of the potential of these compounds as therapeutic agents in the treatment of Parkinson's disease, although physicochemical properties may require optimisation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this review, we first highlighted on C-methyl-branched nucleosides and nucleotides approved as anti-hepatitis C infection (HCV) drugs, their mechanism of action and recent progress in the development of new clinical candidates. Then, we report on our attempt to develop several C-methyl nucleosides/tides potentially useful for treatment of various diseases such cancer, pain, epilepsy and glaucoma. Design, synthesis and pharmacological screening of 1'-C-, 2'-C-, 3'-C-methyladenosine or other purine/pyrimidine nucleosides allowed us to discover some promising new molecules. 3'-C-Methyladenosine showed antitumor activity against several human tumor cell lines. We have investigated the mechanism of action of 3;-C-methyladenosine that proved to be an effective inhibitor of ribonucleotide reductase. Moreover, we will also summarize the chemical and biological properties of some of the recent N6-substituted and 5', N6-disubstituted 2'-C-methyladenosine derivatives that were synthetized in our laboratory and evaluated as A1 adenosine receptor agonists. 2-Chloro-2'- C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), 5'-chloro-5'-deoxy-N6-(±)-(endo-norborn- 2-yl)adenosine (5'Cl5'd-(±)-ENBA) and 2'-C-methyl-5'-chloro-5'-deoxy-N6-(±)-(endonorborn- 2-yl)adenosine (2'-Me-5'Cl5'd-(±)-ENBA) displayed high hA1AR affinity and selectivity. 2'-Me-CCPA and 5'Cl5'd-(±)-ENBA showed significant analgesic properties.

Adenosine A1 receptor (A1AR) stimulation is a powerful protective mechanism in cerebral and cardiac ischemia-reperfusion injury. Despite this, therapeutic targeting of the A1AR for the treatment of ischemia-reperfusion injury has been largely unsuccessful, as high concentrations of prototypical A1AR agonists impart significant hemodynamic effects, particularly pronounced bradycardia, atrioventricular block and hypotension. Exploiting the phenomenon of biased agonism to develop ligands that promote A1AR cytoprotection in the absence of adverse hemodynamic effects remains a relatively unexplored, but exciting, approach to overcome current limitations. In native systems, the atypical A1AR agonists VCP746 and capadenoson retain cytoprotective signaling in the absence of bradycardia, a phenomenon suggestive of biased agonism. The current study used pharmacological inhibitors to investigate A1AR mediated cytoprotective signal transduction in a CHO FlpIn cell background, thus identifying candidate pathways for quantitative bias profiling, including cAMP, extracellular signal-regulated kinases 1 and 2 and Akt1/2/3. Subsequently, effects on cell survival and the bias profile of VCP746 and capadenoson were determined and compared to that of the prototypical A1AR agonists, NECA, R-PIA, MeCCPA and CPA. We found that prototypical agonists do not display significant bias for any of the pathways assessed. In contrast, VCP746 and capadenoson show significant bias away from calcium mobilization relative to all pathways tested. These studies demonstrate that quantitative "fingerprinting" of biased agonism within a model system can enable ligands to be clustered by their bias profile, which in turn may be predictive of preferential physiologically relevant in vivo pharmacology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fragment-based lead discovery (FBLD) holds great promise for drug discovery, but applications to G protein-coupled receptors (GPCRs) have been limited by a lack of sensitive screening techniques and scarce structural information. If virtual screening against homology models of GPCRs could be used to identify fragment ligands, FBLD could be extended to numerous important drug targets and contribute to efficient lead generation. Access to models of multiple receptors may further enable the discovery of fragments that bind specifically to the desired target. To investigate these questions, we used molecular docking to screen >500 000 fragments against homology models of the A3 and A1 adenosine receptors (ARs) with the goal to discover A3AR-selective ligands. Twenty-one fragments with predicted A3AR-specific binding were evaluated in live-cell fluorescence-based assays; of eight verified ligands, six displayed A3/A1 selectivity, and three of these had high affinities ranging from 0.1 to 1.3 μM. Subsequently, structure-guided fragment-to-lead optimization led to the identification of a >100-fold-selective antagonist with nanomolar affinity from commercial libraries. These results highlight that molecular docking screening can guide fragment-based discovery of selective ligands even if the structures of both the target and antitarget receptors are unknown. The same approach can be readily extended to a large number of pharmaceutically important targets.

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

With the aim of finding new adenosine receptor (AR) ligands presenting the 3-amidocoumarin scaffold, a study focusing on the discovery of new chemical entities was carried out. The synthesized compounds 1-8 were evaluated in radioligand binding (A1, A2A and A3) and adenylyl cyclase activity (A2B) assays in order to determine their affinity for human AR subtypes. The 3-benzamide derivative 4 showed the highest affinity of the whole series and was more than 30-fold selective for the A3 AR (Ki=3.24 μM). The current study supported that small structural changes in this scaffold allowed modulating the affinity resulting in novel promising classes of A1, A2A, and/or A3 AR ligands. We also performed docking calculations in hA2A and hA3 to identify the hypothetical binding mode for the most active compounds. In addition, some ADME properties were calculated in order to better understand the potential of these compounds as drug candidates., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Here we report novel thiazole-thiophene conjugates as adenosine receptor antagonists. All the molecules were evaluated for their binding affinity for adenosine receptors. Most of the molecules were found to interact with the A1, A2A and A3 adenosine receptor subtypes with good affinity values. The most potent and selective compound 8n showed an A3Ki value of 0.33μM with selectivity ratios of >90 versus the A1 and >30 versus the A2 subtypes. For compound 8n docking studies into the binding site of the A3 adenosine receptor are provided to visualize its binding mode., (Copyright © 2015. Published by Elsevier Ltd.)

A series of N(6)-substituted-5'-C-(2-ethyl-2H-tetrazol-5-yl)-adenosine and 2-chloro-adenosine derivatives was synthesized as novel, highly potent dual acting hA1AR agonists and hA3AR antagonists, potentially useful in the treatment of glaucoma and other diseases. The best affinity and selectivity profiles were achieved by N(6)-substitution with a 2-fluoro-4-chloro-phenyl- or a methyl- group. Through an in silico receptor-driven approach, the molecular bases of the hA1- and hA3AR recognition and activation of this series of 5'-C-ethyl-tetrazolyl derivatives were explained.

Adenosine is an ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1, A2A, A2B and A3 adenosine receptors (ARs). Allosteric enhancers to A1ARs may represent novel therapeutic agents because they increase the activity of these receptors by mediating a shift to their active form in the A1AR-G protein ternary complex. In this manner, they are able to amplify the action of endogenous adenosine, which is produced in high concentrations under conditions of metabolic stress. A1AR allosteric enhancers could be used as a justifiable alternative to the exogenous agonists that are characterized by receptor desensitization and downregulation. In this review, an analysis of some of the most interesting allosteric modulators of A1ARs has been reported.

A Sonogashira coupling strategy was employed to synthesize a new series of allosteric modulators for the A1 adenosine receptor based on the 2-amino-3-(p-chlorobenzoyl)-4-substituted thiophene skeleton, with a two-carbon (rigid or flexible) linker between the 5-position of the thiophene ring and a (hetero)aryl or alkyl moiety. Among the compounds characterized by the presence of a common phenylacetylene moiety at the 5-position of the thiophene ring, the neopentyl substitution at the 4-position supported a strong activity. In the series of 4-neopentyl derivatives, the presence of an acetylene spacer at the 5-position of the thiophene is optimal for activity, whereas reduction of the acetylene to an ethyl moiety decreased activity, both in functional and binding assays. Derivatives 4e, 4g-h, 4j, 4l, and 4m were the most promising compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor, with 4e as the best compound of the series. The latter compound also retarded the dissociation of another radiolabeled agonist, [(3)H]NECA, from the receptor.

In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl3) enhanced the neurotoxicity of the amyloid beta(25-35) fragment (Abeta(25-35)) in neuroblastoma cells and affected the expression of Alzheimer's disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A2A adenosine receptors (A2AR), although it also inhibits A1Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta(25-35)+AlCl3) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta(25-35)+AlCl3 were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A1R and A2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl3 treatment exacerbated Abeta(25-35) toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta(25-35) only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta(25-35)+AlCl3-induced damage, suggesting that A1Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A1R inhibition in the setting of combined Abeta(25-35)+AlCl3 neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A₃AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of β-D-apio-D-furano- and α-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N(6) of β-D-apio-D-furanoadenosine afforded an A₃AR antagonist (10c, Ki=0.98 μM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki=3.07 μM). The structural basis for this difference was examined by docking to an A₃AR model; the antagonist lacked a crucial interaction with Thr94., (Published by Elsevier Ltd.)

Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that adenosine A₁ receptors (A₁Rs) form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A₁Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that the protomer-protomer interactions in the A₁R homomers account for some of the pharmacological characteristics of agonist and antagonist binding to A₁Rs. These pharmacological properties include the appearance of cooperativity in agonist binding, the change from a biphasic saturation curve to a monophasic curve in self-competition experiments and the molecular cross-talk detected when two different specific molecules bind to the receptor. In this last case, we discovered that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A₁R homomer, a pharmacological characteristic that correlates with the low caffeine concentrations-induced activation of agonist-promoted A₁R signaling. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides were synthesized and tested for their affinity toward A₁, A2A , and A₃ adenosine receptor subtypes. Biological results demonstrated that the introduction of a fluorine atom at the ortho position of the 9-benzyl group generally enhanced affinity toward A₁ subtype and did not significantly affect A2A and A₃ affinity. Very interesting is the compound bearing a meta-fluorophenyl substituent on the carbonyl carbon of the amide group, which shows significantly high A₁/A2A-A₃ selectivity. Compounds of this new series, together with the previously published analogs without the fluorine atom on the 9-benzyl group, constituted the starting dataset for the development of QSAR models. The models obtained were able to rationally describe the affinity trends resulting from biological testing and to enable investigation of the role of different substituents on the 8-azapurine scaffold, as well as the influence of the newly introduced fluorine atom on the benzyl moiety. The said QSAR models can also assist in the design of new compounds selectively active on A₁ adenosine receptors. Furthermore, a molecular docking study was carried out to assess hypothetical binding mode of N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides to A₁ adenosine receptors., (© 2013 John Wiley & Sons A/S.)