Biased agonism at adenosine receptors

Adenosine modulates many aspects of human physiology and pathophysiology through binding to the adenosine family of G protein-coupled receptors, which are comprised of four subtypes, the A₁R, A_2AR, A_2BR and A₃R. Modulation of adenosine receptor function by exogenous agonists, antagonists and allosteric modulators can be beneficial for a number of conditions including cardiovascular disease, Parkinson's disease, and cancer. Unfortunately, many preclinical drug candidates targeting adenosine receptors have failed in clinical trials due to limited efficacy and/or severe on-target undesired effects. To overcome the key barriers typically encountered when transitioning adenosine receptor ligands into the clinic, research efforts have focussed on exploiting the phenomenon of biased agonism. Biased agonism provides the opportunity to develop ligands that favour therapeutic signalling pathways, whilst avoiding signalling associated with on-target undesired effects. Recent studies have begun to define the structure-function relationships that underpin adenosine receptor biased agonism and establish how this phenomenon can be harnessed therapeutically. In this review we describe the recent advancements made towards achieving therapeutically relevant biased agonism at adenosine receptors.     

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions

Adenosine A₁ and A_2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A₁ and A_2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A₁ and A_2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A₁ receptor with K_i values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A_2A receptor. These substitution patterns led to enhanced adenosine A₁ and A_2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A₁ receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A₁ receptors. In conclusion, compounds 3 and 12, showed the best adenosine A₁ and A_2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.     

Enhancer and competitive allosteric modulation model for G-protein-coupled receptors

A new mathematical model, referred to as Enhancer and Competitive Allosteric Modulator (ECAM) model, developed with the aim of quantitatively describing the interaction of an allosteric modulator with both enhancer and competitive properties towards G-protein-coupled receptors is described here. Model simulations for equilibrium (displacement-like and saturation-like), and kinetic (association and dissociation) binding experiments were performed. The results showed the ability of the model to interpret a number of possible ligand-receptor binding behaviors. In particular, the binding properties of PD81723, an enhancer and competitive allosteric modulator for the adenosine A₁ receptor, were experimentally evaluated by radioligand binding assays and interpreted by the ECAM model. The results also offer a theoretical background enabling the design and optimization of compounds endowed with allosteric enhancer, competitive, agonist, antagonist, and inverse agonist properties.     

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A₁ adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A₁, A_2A, A_2B and A₃ adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A₁, A₃ or dual A₁/A₃ adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.     

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1^G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A_2A receptor agonist (CGS21680), antagonist (KW6002) or the A₁ receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A_2A receptors modified the progressive loss of motor skills or survival of mSOD1^G93A mice. Conversely, blockade of adenosine A₁ receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

     

Novel positive allosteric modulators of A2B adenosine receptor acting as bone mineralisation promoters

Small-molecules acting as positive allosteric modulators (PAMs) of the A_2B adenosine receptor (A_2B AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds (4-13) exhibiting different degrees of chemical similarity with three indole derivatives (1-3), which have been recently identified by us as PAMs of the A_2B AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of 4-13 in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative 9, confirmed that such a molecule behaves as PAM of the A_2B AR.     

Synthesis,biological assays and QSAR studies of N-(9-benzyl-2-phenyl-8-azapurin-6-yl)-amides as ligands for A1 adenosine receptors

2-Phenyl-9-benzyl-8-azapurines, bearing at the 6 position an amido group interposed between the 8-azapurine moiety and an alkyl or a substituted phenyl group, have been synthesised and assayed as ligands for adenosine receptors. All the compounds show high affinity for the A₁ adenosine receptor, and many of them also show a good selectivity for A₁ with respect to A_2A and A₃ adenosine receptors. Based on the quite rich library containing such compounds and relevant biological data, QSAR models, able to rationalise the results and to give a quantitative estimate of the observed trends were also developed. The obtained models can assist in the design of new compounds selectively active on A₁ adenosine receptor.     

腺苷和睡眠觉醒调节

腺苷作为神经调质,调节多种神经生物学功能.随觉醒时间延长,动物脑内腺苷水平逐渐增高,在睡眠期显著降低.因此,腺苷被认为是调节睡眠的内稳态因子之一.腺苷受体(receptor,R)有A1R、A2AR、A2BR和A3R四种亚型,其中A1R和A2AR与诱导睡眠相关.激活A1R可抑制促觉醒神经元诱导睡眠,也可抑制促眠神经元导致...     

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A_2A receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A_2B and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A_2B receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A_2A receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A_2A and A₃) receptors by endogenous adenosine. The selective A_2B receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1–10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A_2A receptors. In contrast to 2-Cl-IB MECA, activation of A_2A receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A_2A receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A_2A receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.     

Enhancement of inosine-mediated A2AR signaling through positive allosteric modulation

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15 h) than adenosine (half-life < 10 s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A_2A receptor (A_2AR). Relative to adenosine inosine exhibits a lower affinity towards the A_2AR. Therefore, it is generally believed that inosine is incapable of activating the A_2AR through direct engagement, but indirectly activates the A_2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A_2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A_2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A_2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A_2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A_2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A_2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.     

Limonene, a natural cyclic terpene, is an agonistic ligand for adenosine A(2A) receptors

Limonene is a major aromatic compound in essential oils extracted from citrus rind. The application of limonene, especially in aromatherapy, has expanded significantly, but its potential effects on cellular metabolism have been elusive. We found that limonene directly binds to the adenosine A_2A receptor, which may induce sedative effects. Results from an in vitro radioligand binding assay showed that limonene exhibits selective affinity to A_2A receptors. In addition, limonene increased cytosolic cAMP concentration and induced activation of protein kinase A and phosphorylation of cAMP-response element-binding protein in Chinese hamster ovary cells transfected with the human adenosine A_2A receptor gene. Limonene also increased cytosolic calcium concentration, which can be achieved by the activation of adenosine A_2A receptors. These findings suggest that limonene can act as a ligand and an agonist for adenosine A_2A receptors.     

Adenosine-induced cell death: evidence for receptor-mediated signalling

Adenosine modulates the proliferation, survival and apoptosis of many different cell types, ranging from epithelial, endothelial and smooth muscle cells, to cells of the immune and neural lineages. In this review, we critically discuss the available in vitro and in vivo data which support a role for adenosine in both development-associated apoptosis, and in diseases characterized by either pathologically increased cell death (e.g., ischemia, trauma and aging-associated neurodegeneration) or abnormally reduced spontaneous apoptosis (e.g., cancer). Particular emphasis is given to the possible role of extracellular adenosine receptors, since these may represent novel and attractive molecular targets for the pharmacological modulation of apoptosis. In some instances, adenosine-induced cell death has been demonstrated to require entry of the nucleoside inside cells; however, in many other cases, activation of specific adenosine extracellular receptors has been demonstrated. Of the four G protein-coupled adenosine receptors so far identified, the A_2A and the A₃ receptors have been specifically implicated in modulation of cell death. For the A₃ receptor, results obtained by exposing both cardiomyocytes and brain astrocytes to graded concentrations of selective agonists suggest induction of both cell protection and cell death. Such opposite effects, which likely depend on the degree of receptor activation, may have important therapeutic implications in the pharmacological modulation of cardiac and brain ischemia. For the A_2A receptor, recent intriguing data suggest a specific role in immune cell death and immunosuppression, which may be relevant to both adenosine-deaminase-immunodeficiency syndrome (a pathology characterized by accumulation of adenosine to toxic levels) and in tumors where induction of apoptosis via activation of specific extracellular receptors may be desirable. Finally, preliminary data suggest that, in a similar way to the adenosine-deaminase-immunodeficiency syndrome, the abnormal accumulation of adenosine in degenerative muscular diseases may contribute to muscle cell death. Although the role of adenosine receptors in this effect still remains to be determined, these data suggest that adenosine-induced apoptosis may also represent a novel pathogenic pathway in muscular dystrophies.     

Epac is required for exogenous and endogenous stimulation of adenosine A<Subscript>2B</Subscript> receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation

Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A₂ receptors (A₂Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A₂Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A_2B receptor (A_2BR) subtype. Stimulation of A_2BR exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A_2BR-mediated antifibrotic effects. Thus, A_2BR is one of the potential therapeutic targets against cardiac fibrosis.     

P1 receptors and cytokine secretion

Evidence has accumulated in the last three decades to suggest tissue protection and regeneration by adenosine in multiple different cell types. Adenosine produced in hypoxic or inflamed environments reduces tissue injury and promotes repair by receptor-mediated mechanisms. Among other actions, regulation of cytokine production and secretion by immune cells, astrocytes and microglia (the brain immunocytes) has emerged as a main mechanism at the basis of adenosine effects in diseases characterized by a marked inflammatory component. Many recent studies have highlighted that signalling through A₁ and A_2A adenosine receptors can powerfully prevent the release of pro-inflammatory cytokines, thus inhibiting inflammation and reperfusion injury. However, the activation of adenosine receptors is not invariably protective of tissues, as signalling through the A_2B adenosine receptor has been linked to pro-inflammatory actions which are, at least in part, mediated by increased release of pro-inflammatory cytokines from epithelial cells, astrocytes and fibroblasts. Here, we discuss the multiple actions of P1 receptors on cytokine secretion, by analyzing, in particular, the role of the various adenosine receptor subtypes, the complex reciprocal interplay between the adenosine and the cytokine systems, their pathophysiological significance and the potential of adenosine receptor ligands as new anti-inflammatory agents.     

Cardioprotective effects of adenosine A1 and A 3 receptor activation during hypoxia in isolated rat cardiac myocytes

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A₁ and A₃ receptors (A₁R and A₃R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4–5 days in vitro), to an atmosphere of a N₂ (95%) and CO₂ (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A₁ and A₃ receptor antagonists abolished the protective effects of adenosine. A₁R and A₃R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A₁R agonist, CCPA, was abolished by an A₁R antagonist, DPCPX, and was not affected by an A₃R antagonist, MRS1523. Cardioprotection caused by the A₃R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A₁R and A₃R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A₁ or A₃ adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A₁R and A₃R agonists may have potential as cardioprotective agents against ischemia or heart surgery.     

Adenosine,adenosine receptors and glaucoma: An updated overview

Background

Glaucoma, a leading cause of blindness worldwide, is an optic neuropathy commonly associated with elevated intraocular pressure (IOP). The major goals of glaucoma treatments are to lower IOP and protect retinal ganglion cells. It has been revealed recently that adenosine and adenosine receptors (ARs) have important roles in IOP modulation and neuroprotection.

Scope of review

This article reviews recent studies on the important roles of adenosine and ARs in aqueous humor formation and outflow facility, IOP and retinal neuroprotection.

Major conclusions

Adenosine and several adenosine derivatives increase and/or decrease IOP via A_2A AR. Activation of A₁ AR can reduce outflow resistance and thereby lower IOP, A₃ receptor antagonists prevent adenosine-induced activation of Cl⁻ channels of the ciliary non-pigmented epithelial cells and thereby lower IOP. A₁ and A_2A agonists can reduce vascular resistance and increase retina and optic nerve head blood flow. A₁ agonist and A_2A antagonist can enhance the recovery of retinal function after ischemia attack. Adenosine acting at A₃ receptors can attenuate the rise in calcium and retinal ganglion cells death accompanying P2X(7) receptor activation.

General significance

Evidence suggested that the adenosine system is one of the potential target systems for therapeutic approaches in glaucoma.     

C2-substituted quinazolinone derivatives exhibit A1 and/or A2A adenosine receptor affinities in the low micromolar range

Antagonists of the adenosine receptors (A₁ and A_2A subtypes) are widely researched as potential drug candidates for their role in Parkinson’s disease-related cognitive deficits (A₁ subtype), motor dysfunction (A_2A subtype) and to exhibit neuroprotective properties (A_2A subtype). Previously the benzo-α-pyrone based derivative, 3-phenyl-1H-2-benzopyran-1-one, was found to display both A₁ and A_2A adenosine receptor affinity in the low micromolar range. Prompted by this, the α-pyrone core was structurally modified to explore related benzoxazinone and quinazolinone homologues previously unknown as adenosine receptor antagonists. Overall, the C2-substituted quinazolinone analogues displayed superior A₁ and A_2A adenosine receptor affinity over their C2-substituted benzoxazinone homologues. The benzoxazinones were devoid of A_2A adenosine receptor binding, with only two compounds displaying A₁ adenosine receptor affinity. In turn, the quinazolinones displayed varying degrees of affinity (low micromolar range) towards the A₁ and A_2A adenosine receptor subtypes. The highest A₁ adenosine receptor affinity and selectivity were favoured by methyl para-substitution of phenyl ring B (A₁K_i = 2.50 μM). On the other hand, 3,4-dimethoxy substitution of phenyl ring B afforded the best A_2A adenosine receptor binding (A_2AK_i = 2.81 μM) among the quinazolinones investigated. In conclusion, the quinazolinones are ideal lead compounds for further structural optimization to gain improved adenosine receptor affinity, which may find therapeutic relevance in Parkinson’s disease-associated cognitive deficits and motor dysfunctions as well as exerting neuroprotective properties.     

The Role of Glial Adenosine Receptors in Neural Resilience and the Neurobiology of Mood Disorders

Adenosine receptors were classified into A₁- and A₂-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that are important for neuronal survival and growth. Neuronal resilience is now considered as of pivotal importance in the neurobiology of mood disorders and their treatment. Both sleep deprivation and electroconvulsive therapy, two effective therapeutic measures in mood disorders, are associated with an increase of adenosine and upregulation of adenosine A₁-receptors in the brain. Parameters closely related to adenosine receptor activation such as cerebral metabolic rate and delta power in the sleep EEG provide indirect evidence that adenosinergic signaling may be associated with the therapeutic response to these measures. Thus, neurotrophic effects evoked by adenosine receptors might be important in the mechanism of action of ECT and perhaps also sleep deprivation.     

K(v) 7 (KCNQ) channel openers normalize central 2-deoxyglucose uptake in a mouse model of mania and increase prefrontal cortical and hippocampal serine-9 phosphorylation levels of GSK3β

In the CNS, an antagonistic interaction has been shown between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor–receptor interaction have recently been explored, as the fine tuning of this target (namely the A_2AR/D₂R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer‐based approach to examine the allosteric modulation of the D₂R within the A_2AR/D₂R oligomer and the dependence of this receptor–receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D₂R. Interestingly, we observed a negative allosteric effect of the D₂R agonist quinpirole on A_2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on intracellular loop 3 of the D₂R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor–receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A_2AR/D₂R oligomer in living cells.     

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators

The A_2B adenosine receptor (A_2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A_2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases.Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A_2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A_2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization.In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A_2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A_2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A_2B AR in the control of osteoblast differentiation, bone formation and fracture repair.