Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons.

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A3 Receptor Expression.

The adenosine A3 receptor (A3AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A3AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A3AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A3AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A3AR in pathologies.

Structure-Activity Studies of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators.

We previously reported 1H-imidazo[4,5-c]quinolin-4-amines as A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs). A3AR agonists, but not PAMs, are in clinical trials for inflammatory diseases and liver conditions. We synthesized new analogues to distinguish 2-cyclopropyl antagonist 17 (orthosteric interaction demonstrated by binding and predicted computationally) from PAMs (derivatives with large 2-alkyl/cycloalkyl/bicycloalkyl groups). We predicted PAM binding at a hydrophobic site on the A3AR cytosolic interface. Although having low Caco-2 permeability and high plasma protein binding, hydrophobic 2-cyclohept-4-enyl-N-3,4-dichlorophenyl, MRS7788 18, and 2-heptan-4-yl-N-4-iodophenyl, MRS8054 39, derivatives were orally bioavailable in rat. 2-Heptan-4-yl-N-3,4-dichlorophenyl 14 and 2-cyclononyl-N-3,4-dichlorophenyl 20 derivatives and 39 greatly enhanced Cl-IB-MECA-stimulated [35S]GTPγS binding Emax, with only 12b trending toward decreasing the agonist EC50. A feasible route for radio-iodination at the p-position of a 4-phenylamino substituent suggests a potential radioligand for allosteric site binding. Herein, we advanced an allosteric approach to developing A3AR-activating drugs that are potentially event- and site-specific in action.

Drugs Targeting the A3 Adenosine Receptor: Human Clinical Study Data.

The A3 adenosine receptor (A3AR) is overexpressed in pathological human cells. Piclidenoson and namodenoson are A3AR agonists with high affinity and selectivity to A3AR. Both induce apoptosis of cancer and inflammatory cells via a molecular mechanism entailing deregulation of the Wnt and the NF-κB signaling pathways. Our company conducted phase I studies showing the safety of these 2 molecules. In the phase II studies in psoriasis patients, piclidenoson was safe and demonstrated efficacy manifested in significant improvements in skin lesions. Namodenoson is currently being developed to treat liver cancer, where prolonged overall survival was observed in patients with advanced liver disease and a Child-Pugh B score of 7. A pivotal phase III study in this patient population has been approved by the FDA and the EMA and is currently underway. Namodenoson is also being developed to treat non-alcoholic steatohepatitis (NASH). A Phase IIa study has been successfully concluded and showed that namodenoson has anti-inflammatory, anti-fibrosis, and anti-steatosis effects. A phase IIb study in NASH is currently enrolling patients. In conclusion, A3AR agonists are promising drug candidates in advanced stages of clinical development and demonstrate safety and efficacy in their targeted indications.

A3 adenosine receptor agonist IB-MECA reverses chronic cerebral ischemia-induced inhibitory avoidance memory deficit.

BACKGROUND: Chronic cerebral ischemia (CCI) is a major cause of subcortical ischemic vascular dementia. Here, we examined the neuroprotective action of the A3 adenosine receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) on white matter lesions following CCI. METHODS: A CCI mouse model was established using unilateral common carotid artery occlusion. IB-MECA and 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523), an antagonist of the A3 adenosine receptor, were administered by intraperitoneal injection. The inhibitory avoidance test was used to examine changes in memory performance. Microtubule-associated protein 2 (MAP-2) and neurofilament were assessed by immunohistochemistry, while expressions of phosphorylated extracellular signal-regulated kinase (ERK), ERK, interferon-beta (IFN-ß), and glial fibrillary acidic protein (GFAP) were assessed by western blot assay. RESULTS: The memory retention score was reduced in vehicle-treated mice compared with IB-MECA-treated (p < 0.05) mice. Compared to sham-operated mice, p-ERK, GFAP and IFN-ß were increased, while MAP-2 and neurofilament were reduced in vehicle-treated mice (p < 0.01 for each). IB-MECA reduced ERK phosphorylation (p < 0.01) and GFAP expression (p < 0.05), but upregulated MAP-2 and IFN-ß (p < 0.01 for both), compared with vehicle. MRS1523 suppressed the effects of IB-MECA on the memory deficit, ERK phosphorylation, and on MAP-2, neurofilament, GFAP and IFN-ß levels. CONCLUSION: Our results suggest that A3 adenosine receptor stimulation ameliorates CCI-induced memory deficits, modulates the ERK signaling pathway, preserves MAP-2 and neurofilament expression, regulates GFAP expression, and upregulates the anti-inflammatory cytokine IFN-ß. Thus, the A3 adenosine receptor may be a therapeutic target for treatment of cognitive disorders and cerebral inflammatory diseases.

Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.

Pharmacological Study of A3 Adenosine Receptor agonist (AB Meca) in Xenograft Lung Cancer Model in Mice through In Silico and In Vivo Approach: Targeting TNF-α.

BACKGROUND: Lung cancer is the leading cause of mortality in India. Adenosine Receptor (AR) has emerged as a novel cancer-specific target. A3AR levels are upregulated in various tumor cells, which may mean that the specific AR may act as a biological marker and target specific ligands leading to cell growth inhibition. AIM: Our aim was to study the efficacy of the adenosine receptor agonist, AB MECA, by in silico (molecular docking) and in vitro (human cancer cells in xenografted mice) studies. METHODS: Molecular docking on the AB-meca and TNF-α was performed using AutoDock. A549 Human lung cancer 2 ×106 cells per microliter per mouse injected via intrabronchial route. Rat TNF-α level was assessed by ELISA method. RESULTS: AB Meca's predicted binding energy (beng) with TNF-α was 97.13 kcal/mol, and the compatible docking result of a small molecular inhibitor with TNF-α native ligand beng was 85.76 kcal/mol. In vivo, a single dose of lung cancer cell A549 is being researched to potentiate tumor development. Doxorubicin and A3AR agonist therapies have lowered TNF-alpha levels that were associated with in silico function. The A3AR Agonist showed myeloprotective effects in the groups treated along with doxorubicin. CONCLUSION: AB MECA's higher binding energy (beng) with TNF-α mediated reduction of tumor growth in our lung cancer in vivo model suggested that it may be an effective therapy for lung cancer.

Intraocular implants loaded with A3R agonist rescue retinal ganglion cells from ischemic damage.

Retinal ganglion cell (RGC) loss underlies several conditions which give rise to significant visual compromise, including glaucoma and ischaemic optic neuropathies. Neuroprotection of RGCs is a clinical well-defined unmet need in these diseases, and adenosine A3 receptor (A3R) activation emerges as a therapeutic pharmacological approach to protect RGCs. A porous biodegradable intraocular implant loaded with 2-Cl-IB-MECA (selective A3R agonist) was used as a strategy to protect RGCs. Drug-loaded PCL implants released 2-Cl-IB-MECA for an extended period and the released 2-Cl-IB-MECA limited glutamate-evoked calcium (Ca2+) rise in RGCs. Retinal thinning due to transient ischemia was not prevented by 2-Cl-IB-MECA-PCL implant. However, 2-Cl-IB-MECA-PCL implants decreased retinal cell death, promoted the survival of RGCs, preserved optic nerve structure and anterograde axonal transport. We further demonstrated that 2-Cl-IB-MECA-loaded PCL implants were able to enhance RGC function that was compromised by transient ischemia. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA released from the PCL implant, this can be envisaged a good therapeutic strategy to protect RGCs.

Targeting the A3 adenosine receptor to prevent and reverse chemotherapy-induced neurotoxicities in mice.

Cisplatin is used to combat solid tumors. However, patients treated with cisplatin often develop cognitive impairments, sensorimotor deficits, and peripheral neuropathy. There is no FDA-approved treatment for these neurotoxicities. We investigated the capacity of a highly selective A3 adenosine receptor (AR) subtype (A3AR) agonist, MRS5980, to prevent and reverse cisplatin-induced neurotoxicities. MRS5980 prevented cisplatin-induced cognitive impairment (decreased executive function and impaired spatial and working memory), sensorimotor deficits, and neuropathic pain (mechanical allodynia and spontaneous pain) in both sexes. At the structural level, MRS5980 prevented the cisplatin-induced reduction in markers of synaptic integrity. In-situ hybridization detected Adora3 mRNA in neurons, microglia, astrocytes and oligodendrocytes. RNAseq analysis identified 164 genes, including genes related to mitochondrial function, of which expression was changed by cisplatin and normalized by MRS5980. Consistently, MRS5980 prevented cisplatin-induced mitochondrial dysfunction and decreased signs of oxidative stress. Transcriptomic analysis showed that the A3AR agonist upregulates genes related to repair pathways including NOTCH1 signaling and chromatin modification in the cortex of cisplatin-treated mice. Importantly, A3AR agonist administration after completion of cisplatin treatment resolved cognitive impairment, neuropathy and sensorimotor deficits. Our results highlight the efficacy of a selective A3AR agonist to prevent and reverse cisplatin-induced neurotoxicities via preventing brain mitochondrial damage and activating repair pathways. An A3AR agonist is already in cancer, clinical trials and our results demonstrate management of neurotoxic side effects of chemotherapy as an additional therapeutic benefit.

A3 adenosine receptor agonists containing dopamine moieties for enhanced interspecies affinity.

Following our study of 4'-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A3AR affinity, we incorporated dopamine-related N6 substituents in the full agonist 5'-methylamide series. N6-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed Ki (nM) 0.563 at hA3AR (∼20,000-fold selective) and 1.54 at mA3AR. 2-Alkyl ethers maintained A3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and ß-arrestin 2 (ßarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA3AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA3AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A3AR agonism in vivo.

The Histamine H4 Receptor Participates in the Anti-Neuropathic Effect of the Adenosine A3 Receptor Agonist IB-MECA: Role of CD4+ T Cells.

A3 adenosine receptor (A3AR) agonists have emerged as potent relievers of neuropathic pain by a T cell-mediated production of IL-10. The H4 histamine receptor (H4R), also implicated in pain modulation, is expressed on T cells playing a preeminent role in its activation and release of IL-10. To improve the therapeutic opportunities, this study aimed to verify the hypothesis of a possible cross-talk between A3AR and H4R in the resolution of neuropathic pain. In the mouse model of Chronic Constriction Injury (CCI), the acute intraperitoneal co-administration of the A3AR agonist IB-MECA (0.5 mg/kg) and the H4R agonist VUF 8430 (10 mg/kg), were additive in counteracting mechano-allodynia increasing IL-10 plasma levels. In H4R-/- mice, IB-MECA activity was reduced, lower pain relief and lower modulation of plasma IL-1ß, TNF-α, IL-6 and IL-10 were shown. The complete anti-allodynia effect of IB-MECA in H4R-/- mice was restored after intravenous administration of CD4+ T cells obtained from naïve wild type mice. In conclusion, a role of the histaminergic system in the mechanism of A3AR-mediated neuropathic pain relief was suggested highlighting the driving force evoked by CD4+ T cells throughout IL-10 up-regulation.

Subtle Chemical Changes Cross the Boundary between Agonist and Antagonist: New A3 Adenosine Receptor Homology Models and Structural Network Analysis Can Predict This Boundary.

Distinguishing compounds' agonistic or antagonistic behavior would be of great utility for the rational discovery of selective modulators. We synthesized truncated nucleoside derivatives and discovered 6c (Ki = 2.40 nM) as a potent human A3 adenosine receptor (hA3AR) agonist, and subtle chemical modification induced a shift from antagonist to agonist. We elucidated this shift by developing new hA3AR homology models that consider the pharmacological profiles of the ligands. Taken together with molecular dynamics (MD) simulation and three-dimensional (3D) structural network analysis of the receptor-ligand complex, the results indicated that the hydrogen bonding with Thr943.36 and His2727.43 could make a stable interaction between the 3'-amino group with TM3 and TM7, and the corresponding induced-fit effects may play important roles in rendering the agonistic effect. Our results provide a more precise understanding of the compounds' actions at the atomic level and a rationale for the design of new drugs with specific pharmacological profiles.

Dual Actions of A2A and A3 Adenosine Receptor Ligand Prevents Obstruction-Induced Kidney Fibrosis in Mice.

Kidney fibrosis is the final outcome of chronic kidney disease (CKD). Adenosine plays a significant role in protection against cellular damage by activating four subtypes of adenosine receptors (ARs), A1AR, A2AAR, A2BAR, and A3AR. A2AAR agonists protect against inflammation, and A3AR antagonists effectively inhibit the formation of fibrosis. Here, we showed for the first time that LJ-4459, a newly synthesized dual-acting ligand that is an A2AAR agonist and an A3AR antagonist, prevents the progression of tubulointerstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed on 6-week-old male C57BL/6 mice. LJ-4459 (1 and 10 mg/kg) was orally administered for 7 days, started at 1 day before UUO surgery. Pretreatment with LJ-4459 improved kidney morphology and prevented the progression of tubular injury as shown by decreases in urinary kidney injury molecular-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) excretion. Obstruction-induced tubulointerstitial fibrosis was attenuated by LJ-4459, as shown by a decrease in fibrotic protein expression in the kidney. LJ-4459 also inhibited inflammation and oxidative stress in the obstructed kidney, with reduced macrophage infiltration, reduced levels of pro-inflammatory cytokines, as well as reduced levels of reactive oxygen species (ROS). These data demonstrate that LJ-4459 has potential as a therapeutic agent against the progression of tubulointerstitial fibrosis.

Synthesis and evaluation of adenosine derivatives as A1, A2A, A2B and A3 adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A3 agonists.

A series of adenosine and 2'-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A1, A2A, A2B and A3 adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A3 AR over other ARs was observed for most tested ligands. In particular, 5'-ethylcarbamoyl-N6-(3-phenylpropyl)adenosine (18), N6-(3-phenylpropyl)-2-chloroadenosine (24) and N6-(3-phenylpropyl)adenosine (40) showed nanomolar A3 affinity (Ki 4.5, 6.4 and 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A3 affinity (Ki 206 nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A3 receptor selectivity than the corresponding phenyl analogs: 7vs. 8, 15vs. 16, 17vs. 18.

Optical control of adenosine A3 receptor function in psoriasis.

Psoriasis is a chronic and relapsing inflammatory skin disease lacking a cure that affects approximately 2% of the population. Defective keratinocyte proliferation and differentiation, and aberrant immune responses are major factors in its pathogenesis. Available treatments for moderate to severe psoriasis are directed to immune system causing systemic immunosuppression over time, and thus concomitant serious side effects (i.e. infections and cancer) may appear. In recent years, the Gi protein-coupled A3 receptor (A3R) for adenosine has been suggested as a novel and very promising therapeutic target for psoriasis. Accordingly, selective, and high affinity A3R agonists are known to induce robust anti-inflammatory effects in animal models of autoimmune inflammatory diseases. Here, we demonstrated the efficacy of a selective A3R agonist, namely MRS5698, in preventing the psoriatic-like phenotype in the IL-23 mouse model of psoriasis. Subsequently, we photocaged this molecule with a coumarin moiety to yield the first photosensitive A3R agonist, MRS7344, which in photopharmacological experiments prevented the psoriatic-like phenotype in the IL-23 animal model. Thus, we have demonstrated the feasibility of using a non-invasive, site-specific, light-directed approach to psoriasis treatment.

Efficient G protein coupling is not required for agonist-mediated internalization and membrane reorganization of the adenosine A3 receptor.

Organization of G protein-coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A3 receptor (A3 AR) to that of the wild-type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA-X-BY630) demonstrated that both wild-type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist-mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A3 AR and A3 AR R108A underwent similar agonist-induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit ß-arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild-type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist-stimulated ß-arrestin recruitment and membrane reorganization of the A3 AR.

LJ529 attenuates mast cell-related inflammation via A3R-PKCε-ALDH2 pathway after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Mast cells (MCs) has been recognized as an effector of inflammation or a trigger of inflammatory factors during stroke. LJ529 was reported to attenuate inflammation through a Gi protein-coupled Adenosine A3 receptor (A3R) after ischemia. Here, we aim to study the protective effect and its mechanism of LJ529 in subarachnoid hemorrhage (SAH) rat model for mast cell-related inflammation. METHODS: 155 Sprague-Dawley adult male rats were used in experiments. Endovascular perforation was used for SAH model. Intraperitoneal LJ529 was performed 1 h after SAH. Neurological scores were measured 24 h after SAH. Rotarod and morris water maze tests were evaluated for 21 days after SAH. Mast cell degranulation was assessed with Toluidine blue staining and Chymase/Typtase protein expressions. Mast cell-related inflammation was evaluated using IL-6, TNF-α and MCP-1 protein expressions. MRS1523, inhibitor of GPR18 and ε-V1-2, inhibitor of PKCε were respectively given intraperitoneally (i.p.) 1 h and 30 min before SAH for mechanism studies. Pathway related proteins were investigated with western blot and immunofluorescence staining. RESULTS: Expression of A3R, PKCε increased after SAH. LJ529 treatment attenuated mast cell degranulation and inflammation. Meanwhile, both short-term and long-term neurological functions were improved after LJ529 treatment. Administration of LJ529 resulted in increased expressions of A3R, PKCε, ALDH2 proteins and decreased expressions of Chymase, Typtase, IL-6, TNF-α and MCP-1 proteins. MRS1523 abolished the treatment effects of LJ529 on neurobehavior and protein levels. ε-V1-2 also reversed the outcomes of LJ529 administration through reduction in protein expressions downstream of PKCε. CONCLUSIONS: LJ529 attenuated mast cell-related inflammation through inhibiting degranulation via A3R-PKCε-ALDH2 pathway after SAH. LJ529 may serve as a potential treatment strategy to relieve post-SAH brain injury.

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A3AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in Rag-KO mice. CD4+ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4+ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when cocultured with CD4+ T cells in an IL-10-dependent manner. A3AR action on CD4+ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+ T cells to release IL-10 is required and sufficient evidence for the use of A3AR agonists as therapeutics.

Activation of adenosine A3 receptor reduces early brain injury by alleviating neuroinflammation after subarachnoid hemorrhage in elderly rats.

The incidence of subarachnoid hemorrhage (SAH) and hazard ratio of death increase with age. Overactivation of microglia contributes to brain damage. This study aimed to investigate the effects of A3 adenosine receptors (A3R) activation on neurofunction and microglial phenotype polarization in the context of SAH in aged rats. The A3R agonist (CI-IB-MECA) and antagonist (MRS1523) were used in the SAH model. Microglia were cultured to mimic SAH in the presence or absence of CI-IB-MECA and/or siRNA for A3R. The neurofunction and status of the microglial phenotype were evaluated. The P38 inhibitor SB202190 and the STAT6 inhibitor AS1517499 were used to explore the signaling pathway. The results showed that SAH induced microglia to polarize to the M(LPS) phenotype both in vivo and in vitro. CI-IB-MECA distinctly skewed microglia towards the M(IL-4) phenotype and ameliorated neurological dysfunction, along with the downregulation of inflammatory cytokines. Knockdown of A3R or inhibition of P38 and/or STAT6 weakened the effects of CI-IB-MECA on microglial phenotypic shifting. Collectively, our findings suggest that activation of A3R exerted anti-inflammatory and neuroprotective effects by regulating microglial phenotype polarization through P38/STAT6 pathway and indicated that A3R agonists may be a promising therapeutic options for the treatment of brain injury after SAH.

Activation of Adenosine A3 Receptor Inhibits Microglia Reactivity Elicited by Elevated Pressure.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness, characterized by optic nerve damage and retinal ganglion cell (RGC) death. Elevated intraocular pressure (IOP) is a main risk factor of glaucoma. Neuroinflammation plays an important role in glaucoma. We have been demonstrating that elevated pressure triggers microglia reactivity that contribute to the loss of RGCs. Adenosine, acting on adenosine receptors, is a crucial modulator of microglia phenotype. Microglia express all adenosine receptors. Previously, we demonstrated that the activation of adenosine A3 receptor (A3R) affords protection to the retina, including RGCs, unveiling the possibility for a new strategy for glaucoma treatment. Since microglial cells express A3R, we now studied the ability of a selective A3R agonist (2-Cl-IB-MECA) in controlling microglia reactivity induced by elevated hydrostatic pressure (EHP), used to mimic elevated IOP. The activation of A3R reduced EHP-induced inducible nitric oxide synthase (iNOS) expression, microglia migration and phagocytosis in BV-2 cells. In retinal microglia, proliferation and phagocytosis elicited by EHP were also decreased by A3R activation. This work demonstrates that 2-Cl-IB-MECA, the selective agonist of A3R, is able to hinder microglia reactivity, suggesting that A3R agonists could afford protection against glaucomatous degeneration through the control of neuroinflammation.