Christa E. Müller

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.

Recent developments in adenosine receptor ligands and their potential as novel drugs

Medicinal chemical approaches have been applied to all four of the adenosine receptor (AR) subtypes (A(1), A(2A), A(2B), and A(3)) to create selective agonists and antagonists for each. The most recent class of selective AR ligands to be reported is the class of A(2B)AR agonists. The availability of these selective ligands has facilitated research on therapeutic applications of modulating the ARs and in some cases has provided clinical candidates. Prodrug approaches have been developed which improve the bioavailability of the drugs, reduce side-effects, and/or may lead to site-selective effects. The A(2A) agonist regadenoson (Lexiscan®), a diagnostic drug for myocardial perfusion imaging, is the first selective AR agonist to be approved. Other selective agonists and antagonists are or were undergoing clinical trials for a broad range of indications, including capadenoson and tecadenoson (A(1) agonists) for atrial fibrillation, or paroxysmal supraventricular tachycardia, respectively, apadenoson and binodenoson (A(2A) agonists) for myocardial perfusion imaging, preladenant (A(2A) antagonist) for the treatment of Parkinsons disease, and CF101 and CF102 (A(3) agonists) for inflammatory diseases and cancer, respectively.

Structure of the human P2Y12 receptor in complex with an antithrombotic drug

P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo, which limits our understanding of this receptor family. P2Y12R regulates platelet activation and thrombus formation, and several antithrombotic drugs targeting P2Y12R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor) suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors. Here we report the 2.6 Å resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.

Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids

The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.

CD39/Ectonucleoside Triphosphate Diphosphohydrolase 1 Provides Myocardial Protection During Cardiac Ischemia/Reperfusion Injury

Background— Extracellular adenosine, generated from extracellular nucleotides via ectonucleotidases, binds to specific receptors and provides cardioprotection from ischemia and reperfusion. In the present study, we studied ecto-enzymatic ATP/ADP-phosphohydrolysis by select members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family during myocardial ischemia. Methods and Results— As a first step, we used a murine model of myocardial ischemia and in situ preconditioning and performed pharmacological studies with polyoxometalate 1, a potent E-NTPDase inhibitor (Na6[H2W12O40]). Polyoxometalate 1 treatment increased infarct sizes and abolished beneficial effects of preconditioning. To define relative contributions of distinct E-NTPDases, we investigated transcriptional responses of E-NTPDases 1 to 3 and 8 to preconditioning. We noted robust and selective induction of E-NTPDase 1 (CD39) transcript and protein. Histological analysis of preconditioned myocardium localized CD39 induction to endothelia and myocytes. Cd39−/− mice exhibited larger infarct sizes with ischemia (cd39+/+ 43.0±3.3% versus cd39−/− 52%±1.8; P<0.05), and cardioprotection was abrogated by preconditioning (cd39+/+ 13.3%±1.5 versus cd39−/− 50.5%±2.8; P<0.01). Heightened levels of injury after myocardial ischemia and negligible preconditioning benefits in cd39−/− mice were corrected by infusion of the metabolic product (AMP) or apyrase. Moreover, apyrase treatment of wild-type mice resulted in 43±4.2% infarct size reduction (P<0.01). Conclusions— Taken together, these studies reveal E-NTPDase 1 in cardioprotection and suggest apyrase in the treatment of myocardial ischemia.

Involvement of adenosine A1 and A2A receptors in the motor effects of caffeine after its acute and chronic administration.

The involvement of adenosine A1 and A2A receptors in the motor effects of caffeine is still a matter of debate. In the present study, counteraction of the motor-depressant effects of the selective A1 receptor agonist CPA and the A2A receptor agonist CGS 21680 by caffeine, the selective A1 receptor antagonist CPT, and the A2A receptor antagonist MSX-3 was compared. CPT and MSX-3 produced motor activation at the same doses that selectively counteracted motor depression induced by CPA and CGS 21680, respectively. Caffeine also counteracted motor depression induced by CPA and CGS 21680 at doses that produced motor activation. However, caffeine was less effective than CPT at counteracting CPA and even less effective than MSX-3 at counteracting CGS 21680. On the other hand, when administered alone in habituated animals, caffeine produced stronger motor activation than CPT or MSX-3. An additive effect on motor activation was obtained when CPT and MSX-3 were coadministered. Altogether, these results suggest that the motor-activating effects of acutely administered caffeine in rats involve the central blockade of both A1 and A2A receptors. Chronic exposure to caffeine in the drinking water (1.0 mg/ml) resulted in tolerance to the motor effects of an acute administration of caffeine, lack of tolerance to amphetamine, apparent tolerance to MSX-3 (shift to the left of its ‘bell-shaped’ dose–response curve), and true cross-tolerance to CPT. The present results suggest that development of tolerance to the effects of A1 receptor blockade might be mostly responsible for the tolerance to the motor-activating effects of caffeine and that the residual motor-activating effects of caffeine in tolerant individuals might be mostly because of A2A receptor blockade.

Agonist-bound structure of the human P2Y12 receptor

The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y12R provided useful insights into ligand binding, the agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here we report the structures of the human P2Y12R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283), reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing questions surrounding P2Y12R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Contribution of E-NTPDase1 (CD39) to renal protection from ischemia-reperfusion injury

Previous studies showed increased extracellular nucleotides during renal ischemia‐reperfusion. While nucleotides represent the main source for extracellular adenosine and adenosine signaling contributes to renal protection from ischemia, we hypothesized a role for ecto‐nucleoside‐triphosphate‐diphosphohydro‐lases (E‐NTPDases) in renal protection. We used a model of murine ischemia‐reperfusion and in situ ischemic preconditioning (IP) via a hanging weight system for atraumatic renal artery occlusion. Initial studies with a nonspecific inhibitor of E‐NTPDases (POM‐1) revealed inhibition of renal protection by IP. We next pursued transcriptional responses of E‐NTPDases (E‐NTPDasel‐3, and 8) to renal IP, and found a robust and selective induction of E‐NTPDase1/CD39 transcript and protein. Moreover, based on clearance studies, plasma electrolytes, and renal tubular histology, IP protection was abolished in gene‐targeted mice for cd39 whereas increased renal adenosine content with IP was attenuated. Furthermore, administration of apyrase reconstituted renal protection by IP in cd39−/− mice. Finally, apyrase treatment of wild‐type mice resulted in increased renal adenosine concentrations and a similar degree of renal protection from ischemia as IP treatment. Taken together, these data identify CD39‐dependent nucleotide phosphohydrolysis in renal protection. Moreover, the present studies suggest apyrase treatment as a novel pharmacological approach to renal diseases precipitated by limited oxygen availability.—Grenz, A., Zhang, H., Hermes, M., Eckle, T., Klingel, K., Huang, D. Y., Muller, C. E., Robson, S. C., Osswald, H., Eltzschig, H. K. Contribution of E‐NTPDasel (CD39) to renal protection from ischemia‐reperfusion injury. FASEB J. 21, 2863–2873 (2007)

CD39/ENTPD1 Expression by CD4+Foxp3+ Regulatory T Cells Promotes Hepatic Metastatic Tumor Growth in Mice

BACKGROUND & AIMS Adenosine mediates immune suppression and is generated by the ectonucleotidases CD39 (ENTPD1) and CD73 that are expressed on vascular endothelial cells and regulatory T cells (Tregs). Although tumor-infiltrating immune cells include Foxp3(+) Tregs, it is not clear whether local adenosine generation by Tregs promotes tumor growth in a CD39-dependent manner. In this study, we have examined the effect of CD39 expression by Tregs on effector immune cell responses to hepatic metastases in vivo. METHODS A model of hepatic metastatic cancer was developed with portal vein infusion of luciferase-expressing melanoma B16/F10 cells and MCA38 colon cancer cells in wild-type (wt) and mutant mice null for Cd39. Chimeric mice were generated by bone marrow transplantation (BMT) using Cd39 null or wt C57BL6 donors and irradiated recipient mice. RESULTS We demonstrate that hepatic growth of melanoma metastatic tumors was strongly inhibited in mice with Cd39 null vasculature or in wt mice with circulating Cd39 null bone marrow-derived cells. We show functional CD39 expression on CD4(+)Foxp3(+) Tregs suppressed antitumor immunity mediated by natural killer (NK) cells in vitro and in vivo. Finally, inhibition of CD39 activity by polyoxometalate-1, a pharmacologic inhibitor of nucleoside triphosphate diphosphohydrolase activity, significantly inhibited tumor growth (P < .001). CONCLUSIONS CD39 expression on Tregs inhibits NK activity and is permissive for metastatic growth. Pharmacologic or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy for secondary hepatic malignancies.