Improvement of the sepsis survival rate by adenosine 2a receptor antagonists depends on immune regulatory functions of regulatory T-cells.

Adenosine shows a significant immunosuppressive effect in sepsis via binding to the adenosine 2a receptor (A2aR). Both genetic deletion and pharmacological inhibition of the A2aR may improve survival in sepsis. However, available research on this protective mechanism is quite limited. We used an A2aR antagonist (ZM241385) to treat a cecal ligation and puncture model of normal mice or regulatory T-cell (Treg)-depletion mice and found that the protective effect of ZM241385 is dependent on Tregs. Mechanically, A2aR inactivation was associated with decreased frequencies and reduced function of Foxp3+ Tregs, as evidenced by Foxp3 and CTLA-4 expression and classical effector T-cell proliferative assays, suggesting Treg modulation is a potential protective mechanism against sepsis. Simultaneously, the function and quantity of abdominal neutrophils were improved with ZM241385 treatment. To see if a link exists between them, Tregs and neutrophils were co-cultured, and it was found that ZM241385 blocked the inhibitory effect of Tregs on neutrophils. According to our research, Tregs play a key role in how A2aR antagonists improve sepsis prognosis and bacterial clearance.

Pentoxifylline as a therapeutic option for pre-eclampsia: a study on its placental effects.

BACKGROUND AND PURPOSE: Recently pentoxifylline, a non-selective phosphodiesterase inhibitor and adenosine receptor antagonist, has attracted much interest for the treatment of the increased vascular resistance and endothelial dysfunction in pre-eclampsia. We therefore investigated the placental transfer, vascular effects and anti-inflammatory actions of pentoxifylline in healthy and pre-eclamptic human placentas. EXPERIMENTAL APPROACH: The placental transfer and metabolism of pentoxifylline were studied using ex vivo placenta perfusion experiments. In wire myography experiments with chorionic plate arteries, pentoxifyllines vasodilator properties were investigated, focusing on the cGMP and cAMP pathways and adenosine receptors. Its effects on inflammatory factors were also studied in placental explants. KEY RESULTS: Pentoxifylline transferred from the maternal to foetal circulation, reaching identical concentrations. The placenta metabolized pentoxifylline into its active metabolite lisofylline (M1), which was released into both circulations. In healthy placentas, pentoxifylline potentiated cAMP- and cGMP-induced vasodilation, as well as causing vasodilation by adenosine A1 antagonism and via NO synthase and PKG. Pentoxifylline also reduced inflammatory factors secretion. In pre-eclamptic placentas, we observed that its vasodilator capacity was preserved, however not via NO-PKG but likely through adenosine signalling. Pentoxifylline neither potentiated vasodilation through cAMP and cGMP, nor suppressed the release of inflammatory factors from these placentas. CONCLUSION AND IMPLICATIONS: Pentoxifylline is transferred across and metabolized by the placenta. Its beneficial effects on the NO pathway and inflammation are not retained in pre-eclampsia, limiting its application in this disease, although it could be useful for other placenta-related disorders. Future studies might focus on selective A1 receptor antagonists as a new treatment for pre-eclampsia.

Pathophysiological Role and Medicinal Chemistry of A2A Adenosine Receptor Antagonists in Alzheimer's Disease.

The A2A adenosine receptor is a protein belonging to a family of four GPCR adenosine receptors. It is involved in the regulation of several pathophysiological conditions in both the central nervous system and periphery. In the brain, its localization at pre- and postsynaptic level in striatum, cortex, hippocampus and its effects on glutamate release, microglia and astrocyte activation account for a crucial role in neurodegenerative diseases, including Alzheimer's disease (AD). This ailment is considered the main form of dementia and is expected to exponentially increase in coming years. The pathological tracts of AD include amyloid peptide-ß extracellular accumulation and tau hyperphosphorylation, causing neuronal cell death, cognitive deficit, and memory loss. Interestingly, in vitro and in vivo studies have demonstrated that A2A adenosine receptor antagonists may counteract each of these clinical signs, representing an important new strategy to fight a disease for which unfortunately only symptomatic drugs are available. This review offers a brief overview of the biological effects mediated by A2A adenosine receptors in AD animal and human studies and reports the state of the art of A2A adenosine receptor antagonists currently in clinical trials. As an original approach, it focuses on the crucial role of pharmacokinetics and ability to pass the blood-brain barrier in the discovery of new agents for treating CNS disorders. Considering that A2A receptor antagonist istradefylline is already commercially available for Parkinson's disease treatment, if the proof of concept of these ligands in AD is confirmed and reinforced, it will be easier to offer a new hope for AD patients.

The Medicinal Chemistry of Caffeine.

The purine alkaloid caffeine is the most widely consumed psychostimulant drug in the world and has multiple beneficial pharmacological activities, for example, in neurodegenerative diseases. However, despite being an extensively studied bioactive natural product, the mechanistic understanding of caffeine's pharmacological effects is incomplete. While several molecular targets of caffeine such as adenosine receptors and phosphodiesterases have been known for decades and inspired numerous medicinal chemistry programs, new protein interactions of the xanthine are continuously discovered providing potentially improved pharmacological understanding and a molecular basis for future medicinal chemistry. In this Perspective, we gather knowledge on the confirmed protein interactions, structure activity relationship, and chemical biology of caffeine on well-known and upcoming targets. The diversity of caffeine's molecular activities on receptors and enzymes, many of which are abundant in the CNS, indicates a complex interplay of several mechanisms contributing to neuroprotective effects and highlights new targets as attractive subjects for drug discovery.

Huntington's Disease: A Review of the Known PET Imaging Biomarkers and Targeting Radiotracers.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a CAG expansion mutation in the huntingtin gene. As a result, intranuclear inclusions of mutant huntingtin protein are formed, which damage striatal medium spiny neurons (MSNs). A review of Positron Emission Tomography (PET) studies relating to HD was performed, including clinical and preclinical data. PET is a powerful tool for visualisation of the HD pathology by non-invasive imaging of specific radiopharmaceuticals, which provide a detailed molecular snapshot of complex mechanistic pathways within the brain. Nowadays, radiochemists are equipped with an impressive arsenal of radioligands to accurately recognise particular receptors of interest. These include key biomarkers of HD: adenosine, cannabinoid, dopaminergic and glutamateric receptors, microglial activation, phosphodiesterase 10 A and synaptic vesicle proteins. This review aims to provide a radiochemical picture of the recent developments in the field of HD PET, with significant attention devoted to radiosynthetic routes towards the tracers relevant to this disease.

Modifications on the Amino-3,5-dicyanopyridine Core To Obtain Multifaceted Adenosine Receptor Ligands with Antineuropathic Activity.

A new series of amino-3,5-dicyanopyridines (1-31) was synthesized and biologically evaluated in order to further investigate the potential of this scaffold to obtain adenosine receptor (AR) ligands. In general, the modifications performed have led to compounds having high to good human (h) A1AR affinity and an inverse agonist profile. While most of the compounds are hA1AR-selective, some derivatives behave as mixed hA1AR inverse agonists/A2A and A2B AR antagonists. The latter compounds (9-12) showed that they reduce oxaliplatin-induced neuropathic pain by a mechanism involving the alpha7 subtype of nAchRs, similar to the nonselective AR antagonist caffeine, taken as the reference compound. Along with the pharmacological evaluation, chemical stability of methyl 3-(((6-amino-3,5-dicyano-4-(furan-2-yl)pyridin-2-yl)sulfanyl)methyl)benzoate 10 was assessed in plasma matrices (rat and human), and molecular modeling studies were carried out to better rationalize the available structure-activity relationships.

Effect of Adenosine and Adenosine Receptor Antagonists on Retinal Müller Cell Inwardly Rectifying Potassium Channels under Exogenous Glutamate Stimulation.

The vitreousness of glaucoma subjects contains elevated glutamate, and excessive extracellular glutamate is toxic to retinal neurons. Therefore, glutamate clearance is potentially impaired in the retina of glaucoma subjects. Müller cells play an important role in maintaining low extracellular levels of neurotransmitters, such as glutamate. A better understanding of the cross-talk between adenosine and glutamate may provide a better characterization of the regulatory network in Müller cells. Here, Müller cells were purified from the rat retina on postnatal day 5 using the papain digestion method. Application of increasing concentrations of glutamate (0-20 mmol/L) caused a dose-dependent decrease in the expression levels of Kir4.1, Kir2.1, GLAST, and GS. Exogenous adenosine regulated Kir channels and subsequently promoted GLAST and GS expression levels in Müller cells under exogenous glutamate stimulation. These effects were partly dependent on adenosine receptors.

Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode.

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A2A and A1ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A2A/A3, ARs, or A3AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A2A/A3, A1/A3, A1/A2A/A3, and A3). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A3 receptor.

Adenosine Receptor Ligands on Cancer Therapy: A Review of Patent Literature.

BACKGROUND: Adenosine is a purine, with an adenine group and a ribose sugar, formed endogenously by ATP catabolism both intracellularly and extracellularly. Among the medicinal features of adenosine and its receptors (A1, A2A, A2B and A3), anticancer activity has been an intense field of research. The anticancer potential of adenosine receptor ligands has been brought to the forefront of research and evidenced in innumerous research articles and patents. OBJECTIVE: The present review focuses on the patent literature from 2002 onwards (2002-May 2017). METHODS: Patents were searched and downloaded from the open access patent data bases and are available online. RESULTS: A significant number of patents (65) have been published on adenosine receptor ligands claiming anticancer activity, or presenting new methods of preparation or treatment thereof, from 2002-2017 (May). From these, 35 were published highlighting the promising attributes of compounds/ methods to fight cancer. Most of the compounds act as adenosine A3 receptor agonists, while others act as antagonists for the other adenosine receptor subtypes. The signaling events triggered by activation of adenosine A3 receptor or by blockade of adenosine A1, A2A and A2B receptors can reverse an environment from being pro-cancer to an anti-cancer in the body. CONCLUSION: The promising anticancer effects mediated by adenosine receptor ligands put them in the forefront as new drug candidates. The present compilation can be worthy to medicinal chemists, pharmacologists, biochemists and other researchers focusing on the putative anticancer activity of adenosine receptor ligands.

Caffeine inhibits hypothalamic A1R to excite oxytocin neuron and ameliorate dietary obesity in mice.

Caffeine, an antagonist of the adenosine receptor A1R, is used as a dietary supplement to reduce body weight, although the underlying mechanism is unclear. Here, we report that adenosine level in the cerebrospinal fluid, and hypothalamic expression of A1R, are increased in the diet-induced obesity (DIO) mouse. We find that mice with overexpression of A1R in the neurons of paraventricular nucleus (PVN) of the hypothalamus are hyperphagic, have glucose intolerance and high body weight. Central or peripheral administration of caffeine reduces the body weight of DIO mice by the suppression of appetite and increasing of energy expenditure. We also show that caffeine excites oxytocin expressing neurons, and blockade of the action of oxytocin significantly attenuates the effect of caffeine on energy balance. These data suggest that caffeine inhibits A1Rs expressed on PVN oxytocin neurons to negatively regulate energy balance in DIO mice.

The Influence of the 1-(3-Trifluoromethyl-Benzyl)-1H-Pyrazole-4-yl Moiety on the Adenosine Receptors Affinity Profile of Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidine Derivatives.

A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A2B adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA2B AR but they displayed affinity at the hA3 AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA3 AR Ki = 11 nM) and selectivity (A1/A3 and A2A/A3 > 9090; A2B/A3 > 909) at the hA3 AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs.

Novel thiazole-thiophene conjugates as adenosine receptor antagonists: synthesis, biological evaluation and docking studies.

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.

Aminophylline, administered at usual doses for rodents in pharmacological studies, induces hippocampal neuronal cell injury under low tidal volume hypoxic conditions in guinea-pigs.

OBJECTIVES: To establish whether aminophylline, administered at usual doses for rodents in pharmacological studies, induces brain injury in systemic hypoxaemia in guinea-pigs. METHODS: A hypoxaemia (partial oxygen tension of arterial blood (PaO2) = 40-60 mmHg) model was developed by low tidal volume mechanical ventilation in guinea-pigs. KEY FINDINGS: Under hypoxic conditions, aminophylline significantly increased the concentration of brain-specific creatine kinase in the serum in a dose- and time-dependent manner. A reduced number of hippocampal neuronal cells in the CA1 region, an increase in the concentration of neuron-specific enolase (NSE) in cerebrospinal fluid (CSF), an increase in lipid hydroperoxides and a decrease in the ratio of glutathione to glutathione disulfide in the brain tissues were also observed. These effects were not observed when aminophylline at the same doses was administered under normoxic conditions (PaO2 = 80-100 mmHg). There was no difference in either serum or CSF concentrations of theophylline between normoxic and hypoxic conditions. Another methylxanthine, caffeine, did not increase the concentration of NSE in CSF. CONCLUSIONS: Aminophylline potentially induces brain damage under hypoxic conditions. We suggest that aminophylline treatment has adverse effects in patients with hypoxaemia subsequent to respiratory disorders such as asthma.

Fluorescent ligands for adenosine receptors.

Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.

Adenosine derived from Staphylococcus aureus-engulfed macrophages functions as a potent stimulant for the induction of inflammatory cytokines in mast cells.

In this study, we attempted to isolate novel mast cell-stimulating molecules from Staphylococcus aureus. Water-soluble extract of S. aureus cell lysate strongly induced human interleukin- 8 in human mast cell line-1 and mouse interleukin-6 in mouse bone marrow-derived mast cells. The active molecule was purified to homogeneity through a C(18) reverse phase HPLC column. By determination of its structure by MALDITOF and (1)H- and (13)C-NMR, adenosine was revealed to be responsible for the observed cytokine induction activities. Further studies using 8-sulfophenyl theophylline, a selective adenosine receptor blocker, verified that purified adenosine can induce interleukin-8 production via adenosine receptors on mast cells. Moreover, adenosine was purified from S. aureusengulfed RAW264.7 cells, a murine macrophage cell line, used to induce phagocytosis of S. aureus. These results show a novel view of the source of exogenous adenosine in vivo and provide a mechanistic link between inflammatory disease and bacterial infection.

Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: developments in understanding the receptor stereoselectivity.

A(1) adenosine receptor antagonists have been proposed to possess an interesting range of potential therapeutic applications. We have already reported the synthesis and the biological characterization of a family of pyrazolo[3,4-b]pyridine derivatives as A(1) adenosine ligands endowed with an antagonistic profile. In the present work, we report the LC separation of enantiomers of our most active A(1) antagonists together with the determination of their absolute configuration by means of X-ray crystal structure analysis. Biological assays confirmed a different activity for the two enantiomers, with the R one showing the higher human A(1)AR affinity. We also developed a homology model of this receptor subtype in order to suggest a binding disposition of the ligands into the hA(1)AR. All of the obtained data suggest that the compound's chirality plays a key role in A(1) affinity.

The role of extracellular adenosine in chemical neurotransmission in the hippocampus and Basal Ganglia: pharmacological and clinical aspects.

Now there is general agreement that the purine nucleoside adenosine is an important neuromodulator in the central nervous system, playing a crucial role in neuronal excitability and synaptic/non-synaptic transmission in the hippocampus and basal ganglia. Adenosine is derived from the breakdown of extra- or intracellular ATP and is released upon a variety of physiological and pathological stimuli from neuronal and non-neuronal sources, i.e. from glial cells and exerts effects diffusing far away from release sites. The resultant elevation of adenosine levels in the extracellular space reaches micromolar level, and leads to the activation A(1), A(2A), A(2B) and A(3) receptors, localized to pre- and postsynaptic as well as extrasynaptic sites. Activation of presynaptic A(1) receptors inhibits the release of the majority of transmitters including glutamate, acetylcholine, noradrenaline, 5-HT and dopamine, whilst the stimulation of A(2A) receptors facilitates the release of glutamate and acetylcholine and inhibits the release of GABA. These actions underlie modulation of neuronal excitability, synaptic plasticity and coordination of neural networks and provide intriguing target sites for pharmacological intervention in ischemia and Parkinson's disease. However, despite that adenosine is also released during ischemia, A(1) adenosine receptors do not participate in the modulation of excitotoxic glutamate release, which is nonsynaptic and is due to the reverse operation of transporters. Instead, extrasynaptic A(1) receptors might be responsible for the neuroprotection afforded by A(1) receptor activation.

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 Parkinson's disease, and CF101 and CF102 (A(3) agonists) for inflammatory diseases and cancer, respectively.

Adenosine receptor modeling: what does the A2A crystal structure tell us?

For a long time, there have been no experimentally determined structural data for any adenosine receptor (AR) and the only approach available for making structure/function correlations about these proteins has been homology modeling. While the early attempts to model these receptors followed the crystallization of bacteriorhodopsin, the cryo-microscopy studies of bovine and frog rhodopsin, and the modeling of a Calpha-template for the TM helices in the rhodopsin family of GPCRs, the crystallization of bovine rhodopsin by Palczewski was of extreme importance as it first provided the crystal structure of an eukaryotic GPCR to be used as template for more realistic homology models. Since then, rhodopsin-based modeling became the routine approach to develop AR structural models that proved to be useful for interpretation of site-directed mutagenesis data and for molecular docking studies. The recently reported crystal structures of the adrenergic beta1 and beta2 receptors only partially confirmed the structural features showed by bovine rhodopsin, raising a question about which template would have been better for further modeling of ARs. Such question remained actually not-answered, due to the publication in late 2008 of the crystal structure of human adenosine A(2A) receptor (AA(2A)R). Since its publication, this structure has been used for ligands docking analysis and has provided a high similarity template for homology modeling of the other AR subtypes. Still, the AA(2A)R crystal structure allows to verify the hypotheses that were made on the basis of the previously reported homology modeling and molecular docking.