A2A adenosine receptor agonist reduced MMP8 expression in healthy M2-like macrophages but not in macrophages from ankylosing spondylitis patients.

BACKGROUND: Ankylosing spondylitis (AS) is an inflammatory autoimmune disease that mostly affects different joints of the body. Macrophages are the predominant cells that mediate disease progression by secreting several pro-inflammatory mediators. Different receptors are involved in macrophages' function including the adenosine receptors (AR). Our main objective in this study was to assess the effect of applying A2A adenosine receptor agonist (CGS-21,680) on the gene expression of inflammatory mediators including bone morphogenetic proteins (BMP)-2, 4 and matrix metalloproteinases (MMP)-3, 8, 9, and 13 on the macrophages from AS patients compared to healthy macrophages. METHODS: Monocytes were isolated from the whole blood of 28 individuals (AS patients and healthy controls in a 1:1 ratio). Macrophages were differentiated using macrophage colony-stimulating factor (M-CSF), and flow cytometry was performed to confirm surface markers. CGS-21,680 was used to treat cells that had been differentiated. Using SYBR green real-time PCR, relative gene expression was determined. RESULTS: Activating A2AAR diminished MMP8 expression in healthy macrophages while it cannot reduce MMP8 expression in patients' macrophages. The effect of A2AAR activation on the expression of BMP2 and MMP9 reached statistical significance neither in healthy macrophages nor in the patients' group. We also discovered a significant positive connection between MMP8 expression and patient scores on the Bath ankylosing spondylitis functional index (BASFI). CONCLUSION: Due to the disability of A2AAR activation in the reduction of MMP8 expression in patients' macrophages and the correlation of MMP8 expression with BASFI index in patients, these results represent defects and dysregulations in the related signaling pathway in patients' macrophages.

A1 Adenosine Receptor Activation Inhibits P2X3 Receptor-Mediated ATP Currents in Rat Dorsal Root Ganglion Neurons.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration dependently suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response to α,ß-meATP, as shown a downward shift of the concentration-response curve for α,ß-meATP. CPA suppressed ATP currents in a voltage-independent manner. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Moreover, CPA suppressed the membrane potential depolarization and action potential bursts, which were induced by α,ß-meATP in DRG neurons. Finally, CPA relieved α,ß-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors. These results indicated that CPA inhibited the activity of P2X3 receptors in rat primary sensory neurons by activating A1 adenosine receptors and its downstream cAMP signaling pathway, revealing a novel peripheral mechanism underlying its analgesic effect.

Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy.

Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.

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.

A single peri-sciatic nerve administration of the adenosine 2A receptor agonist ATL313 produces long-lasting anti-allodynia and anti-inflammatory effects in male rats.

Neuropathic pain is a widespread problem which remains poorly managed by currently available therapeutics. Peripheral nerve injury and inflammation leads to changes at the nerve injury site, including activation of resident and recruited peripheral immune cells, that lead to neuronal central sensitization and pain amplification. The present series of studies tested the effects of peri-sciatic nerve delivery of single doses of adenosine 2A receptor (A2aR) agonists on pain and neuroinflammation. The data provide converging lines of evidence supportive that A2aR agonism at the site of peripheral nerve injury and inflammation is effective in suppressing ongoing neuropathic pain. After A2aR agonism resolved neuropathic pain, a return of pain enhancement (allodynia) was observed in response to peri-sciatic injection of H-89, which can inhibit protein kinase A, and by peri-sciatic injection of neutralizing antibody against the potent anti-inflammatory cytokine interleukin-10. A2aR agonist actions at the nerve injury site suppress neuroinflammation, as reflected by decreased release of interleukin-1ß and nitric oxide, as well as decreased sciatic expression of markers of monocytes/macrophages and inducible nitric oxide synthase. Taken together, the data are supportive that A2aR agonists, acting at the level of peripheral nerve injury, may be of therapeutic value in treating chronic pain of neuroinflammatory origin.

How to influence the mesenchymal stem cells fate? Emerging role of ectoenzymes metabolizing nucleotides.

Extracellular purines, principally adenosine triphosphate and adenosine, are among the oldest evolutionary and widespread chemical messengers. The integrative view of purinergic signaling as a multistage coordinated cascade involves the participation of nucleotides/nucleosides, their receptors, enzymes metabolizing extracellular nucleosides and nucleotides as well as several membrane transporters taking part in the release and/or uptake of these molecules. In view of the emerging data, it is evident and widely accepted that an extensive network of diverse enzymatic activities exists in the extracellular space. The enzymes regulate the availability of nucleotide and adenosine receptor agonists, and consequently, the course of signaling events. The current data indicate that mesenchymal stem cells (MSCs) and cells induced to differentiate exhibit different sensitivity to purinergic ligands as well as a distinct activity and expression profiles of ectonucleotidases than mature cells. In the proposed review, we postulate for a critical role of these enzymatic players which, by orchestrating a fine-tune regulation of nucleotides concentrations, are integrally involved in modulation and diversification of purinergic signals. This specific hallmark of the MSC purinome should be linked with cell-specific biological potential and capacity for tissue regeneration. We anticipate this publication to be a starting point for scientific discussion and novel approach to the in vitro and in vivo regulation of the MSC properties.

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.

A New Class of Fluorinated A2A Adenosine Receptor Agonist with Application to Last-Step Enzymatic [18 F]Fluorination for PET Imaging.

The A2A adenosine receptor belongs to a family of G-coupled protein receptors that have been subjected to extensive investigation over the last few decades. Due to their prominent role in the biological functions of the heart, lungs, CNS and brain, they have become a target for the treatment of illnesses ranging from cancer immunotherapy to Parkinson's disease. The imaging of such receptors by using positron emission tomography (PET) has also been of interest, potentially providing a valuable tool for analysing and diagnosing various myocardial and neurodegenerative disorders, as well as offering support to drug discovery trials. Reported herein are the design, synthesis and evaluation of two new 5'-fluorodeoxy-adenosine (FDA)-based receptor agonists (FDA-PP1 and FDA-PP2), each substituted at the C-2 position with a terminally functionalised ethynyl unit. The structures enable a synthesis of 18 F-labelled analogues by direct, last-step radiosynthesis from chlorinated precursors using the fluorinase enzyme (5'-fluoro-5'-deoxyadenosine synthase), which catalyses a transhalogenation reaction. This delivers a new class of A2A adenosine receptor agonist that can be directly radiolabelled for exploration in PET studies.

How does adenosine control neuronal dysfunction and neurodegeneration?

The adenosine modulation system mostly operates through inhibitory A1 (A1 R) and facilitatory A2A receptors (A2A R) in the brain. The activity-dependent release of adenosine acts as a brake of excitatory transmission through A1 R, which are enriched in glutamatergic terminals. Adenosine sharpens salience of information encoding in neuronal circuits: high-frequency stimulation triggers ATP release in the 'activated' synapse, which is locally converted by ecto-nucleotidases into adenosine to selectively activate A2A R; A2A R switch off A1 R and CB1 receptors, bolster glutamate release and NMDA receptors to assist increasing synaptic plasticity in the 'activated' synapse; the parallel engagement of the astrocytic syncytium releases adenosine further inhibiting neighboring synapses, thus sharpening the encoded plastic change. Brain insults trigger a large outflow of adenosine and ATP, as a danger signal. A1 R are a hurdle for damage initiation, but they desensitize upon prolonged activation. However, if the insult is near-threshold and/or of short-duration, A1 R trigger preconditioning, which may limit the spread of damage. Brain insults also up-regulate A2A R, probably to bolster adaptive changes, but this heightens brain damage since A2A R blockade affords neuroprotection in models of epilepsy, depression, Alzheimer's, or Parkinson's disease. This initially involves a control of synaptotoxicity by neuronal A2A R, whereas astrocytic and microglia A2A R might control the spread of damage. The A2A R signaling mechanisms are largely unknown since A2A R are pleiotropic, coupling to different G proteins and non-canonical pathways to control the viability of glutamatergic synapses, neuroinflammation, mitochondria function, and cytoskeleton dynamics. Thus, simultaneously bolstering A1 R preconditioning and preventing excessive A2A R function might afford maximal neuroprotection. The main physiological role of the adenosine modulation system is to sharp the salience of information encoding through a combined action of adenosine A2A receptors (A2A R) in the synapse undergoing an alteration of synaptic efficiency with an increased inhibitory action of A1 R in all surrounding synapses. Brain insults trigger an up-regulation of A2A R in an attempt to bolster adaptive plasticity together with adenosine release and A1 R desensitization; this favors synaptotocity (increased A2A R) and decreases the hurdle to undergo degeneration (decreased A1 R). Maximal neuroprotection is expected to result from a combined A2A R blockade and increased A1 R activation. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Naturally occurring N(6)-substituted adenosines (cytokinin ribosides) are in vitro inhibitors of platelet aggregation: an in silico evaluation of their interaction with the P2Y(12) receptor.

A few naturally occurring N(6)-substituted adenosine derivatives (cytokinin ribosides) were investigated as inhibitors of platelet aggregation induced in vitro by collagen and their activity range was demonstrated (IC50: 6.77-141 µM). A docking study suggests that anti-aggregation activity of these compounds could involve an interaction with the P2Y12 receptor binding site.

Protective effect of adenosine receptors against lipopolysaccharide-induced acute lung injury.

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) affect 200,000 people a year in the USA. Pulmonary vascular and specifically endothelial cell (EC) barrier compromise is a hallmark of these diseases. We have recently shown that extracellular adenosine enhances human pulmonary (EC) barrier via activation of adenosine receptors (ARs) in cell cultures. On the basis of these data, we hypothesized that activation of ARs might exert barrier-protective effects in a model of ALI/ARDS in mice. To test this hypothesis, we examined the effects of pre- and posttreatment of adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective stable AR agonist, on LPS-induced lung injury. Mice were given vehicle or LPS intratracheally followed by adenosine, NECA, or vehicle instilled via the internal jugular vein. Postexperiment cell counts, Evans Blue Dye albumin (EBDA) extravasation, levels of proteins, and inflammatory cytokines were analyzed. Harvested lungs were used for histology and myeloperoxidase studies. Mice challenged with LPS alone demonstrated an inflammatory response typical of ALI. Cell counts, EBDA extravasation, as well as levels of proteins and inflammatory cytokines were decreased in adenosine-treated mice. Histology displayed reduced infiltration of neutrophils. NECA had a similar effect on LPS-induced vascular barrier compromise. Importantly, posttreatment with adenosine or NECA recovers lung vascular barrier and reduces inflammation induced by LPS challenge. Furthermore, adenosine significantly attenuated protein degradation of A2A and A3 receptors induced by LPS. Collectively, our results demonstrate that activation of ARs protects and restores vascular barrier functions and reduces inflammation in LPS-induced ALI.

Antidepressant effects of sleep deprivation require astrocyte-dependent adenosine mediated signaling.

Major depressive disorder is a debilitating condition with a lifetime risk of ten percent. Most treatments take several weeks to achieve clinical efficacy, limiting the ability to bring instant relief needed in psychiatric emergencies. One intervention that rapidly alleviates depressive symptoms is sleep deprivation; however, its mechanism of action is unknown. Astrocytes regulate responses to sleep deprivation, raising the possibility that glial signaling mediates antidepressive-like actions of sleep deprivation. Here, we found that astrocytic signaling to adenosine (A1) receptors was required for the robust reduction of depressive-like behaviors following 12 hours of sleep deprivation. As sleep deprivation activates synaptic A1 receptors, we mimicked the effect of sleep deprivation on depression phenotypes by administration of the A1 agonist CCPA. These results provide the first mechanistic insight into how sleep deprivation impacts mood, and provide a novel pathway for rapid antidepressant development by modulation of glial signaling in the brain.

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.

Antrodia cinnamomea exhibits a potent neuroprotective effect in the PC12 Cell-Aß25-35 model - pharmacologically through adenosine receptors and mitochondrial pathway.

Antrodia cinnamomea has a diversity of therapeutic effects including anticancer properties. Its neuroprotective effect is rarely cited. We hypothesized that due to its high phenol, triterpenoid, and adenosine contents, it might exhibit a potent neuroprotective effect. The PC12 cell model was used to investigate its pharmaceutical effects. Congo red staining was used to identify the activation of Aß25-35. Chemical analysis indicated that the ethanolic extract of Antrodia cinnamomea contained a huge amount (mg/g ethanolic extract of Antrodia cinnamomea) of polyphenolics (133 ± 7), flavonoids (114 ± 6), triterpenoids (175 ± 26), and adenosine (370 ± 17). When tested with Aß25-35 (15 µM), the cell viability was suppressed in a dose-dependent fashion with an IC50 value of 10 µM. The biochemical parameters upregulated by Aß25-35 (15 µM) involved TNF-α, ROS, MDA, NO, and the intracellular calcium ions. These adverse effects were effectively ameliorated by the ethanolic extract of Antrodia cinnamomea (1 µg/mL). The Western blot analysis revealed that Aß25-35 downregulated BcL-2/Bax and upregulated cleaved caspases-9 and - 3 without affecting cleaved caspase-8. The G2/M arrest elicited by Aß25-35 was ameliorated by the ethanolic extract of Antrodia cinnamomea. TUNEL assay confirmed the apoptosis, and the ethanolic extract of Antrodia cinnamomea downregulated adenosine A1 and adenosine A2A receptors. Taken together, Aß25-35 tends to induce neurotoxicity on PC12 cells. The ethanolic extract of Antrodia cinnamomea is capable of suppressing its neurotoxicity by rescuing the mitochondrial apoptosis pathway and simultaneously by downregulating adenosine A1 and adenosine A2A receptors to retard neurodegeneration and memory dysfunction.

Cytotoxic purine nucleoside analogues bind to A1, A2A, and A3 adenosine receptors.

Fludarabine, clofarabine, and cladribine are anticancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3)). Radioligand binding studies utilizing Chinese hamster ovary cells, stably transfected with adenosine A(1), A(2A), or A(3) receptor subtype, were used to assess the binding affinities of these compounds, whereas adenylyl cyclase activity was used to assess the binding to A(2B) receptors. Clofarabine and cladribine both bound to the A(2A) receptor with a K (i) of 17 and 15 µM, respectively. Clofarabine was the only adenosine analogue to bind to the A(3) receptor with a K (i) of 10 µM, and none of these compounds bound to the A(2B) receptor. Results show that clofarabine, cladribine, and fludarabine bind to the A(1) receptor. In addition, clofarabine, cladribine, and fludarabine were A(1) agonists (IC(50) 3.1, 30, and 30 µM, respectively). Neither pyrimidine nucleoside analogues gemcitabine nor cytarabine associated with any of the adenosine receptor subtypes (K (i) > 100µM). This is the first report of an interaction between all adenosine receptor subtypes and chemotherapeutic nucleoside analogues commonly used in the treatment of cancer. Therefore, activation of these receptors may be at least one mechanism through which fludarabine-associated toxicity occurs.

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.

Differential trafficking of adenosine receptors in hippocampal neurons monitored using GFP- and super-ecliptic pHluorin-tagged receptors.

Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischaemia. To address this we examined the agonist-dependent internalisation of C-terminal GFP-tagged A(1)Rs, A(2A)Rs and A(3)Rs in primary hippocampal neurons. Furthermore, we developed a novel super-ecliptic pHluorin (SEP)-tagged A(1)R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A(1)Rs in real-time. We demonstrate the differential trafficking of ARs in neurons: A(3)Rs internalise more rapidly than A1Rs, with little evidence of appreciable A(2A)R trafficking over the time-course of the experiments. Furthermore, the novel SEP-A(1)R construct revealed the time-course of internalisation and recovery of cell-surface expression to occur within minutes of agonist exposure and removal, respectively. These observations highlight the labile nature of A(1)R and A(3)Rs when expressed at the neuronal plasma membrane. Given the high levels of adenosine in the brain during ischaemia and seizures, internalisation of the inhibitory A(1)R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states.

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.

Allosteric modulators for adenosine receptors: an alternative to the orthosteric ligands.

Adenosine is a ubiquitous homeostatic substance which exerts its action by triggering four different cell membrane G protein-coupled receptors, classified as A(1), A(2A), A(2B), and A(3). Being widely distributed and deeply involved in several physiological functions, as well as pathological disorders, these receptors represent an excellent drug target and the development of specific ligands has been tested as a promising therapeutic concept. Among the obtainable ligands, allosteric modulators offer higher advantages with respect to classical orthosteric compounds, as they possible to achieve greater selectivity and better modulatory control at disease mediating receptors. Actually, synergizing with adenosine bound to the primary binding site, these compounds may modify receptor functions through interaction with an additional binding site. As a consequence, their actions depend directly on the release of the endogenous agonist. A number of compound have been developed as effective allosteric modulators. Most of them target adenosine A(1) and A(3) receptor subtypes as, to date, little or no research attempt have been made to improve the field of A(2A) and A(2B) ligands. This review updates literature on the allosteric modulators that has appeared in the last few years, focusing its attention on medicinal chemistry, in terms of chemical structure and structure-activity relationships. This will provide new perspectives on existing data and an exciting starting point for the development of novel and more effective modulators.