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Drugs targeting adenosine receptors (AR) can provide treatment for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4

A3 adenosine receptor (A3AR) is a cell membrane protein, which has been found to be overexpressed in a large number of cancer types. This receptor plays an important role in cancer by interacting with adenosine. Specifically, A3AR has a dual nature in different pathophysiological conditions, as it is expressed according to tissue type and stimulated by an adenosine dose-dependent manner. A3AR activation leads to tumor growth, cell proliferation and survival in some cases, while triggering cytostatic and apoptotic pathways in others. This review aims to describe the most relevant aspects of A3AR activation and its ligands whereas it summarizes A3AR activities in cancer. Progress in the field of A3AR modulators, with a potential therapeutic role in cancer treatment are reported, as well.

The adenosine A

Adenosine is an important neuromodulator in the CNS, regulating neuronal survival and synaptic transmission. The antioxidant ascorbate (the reduced form of vitamin C) is concentrated in CNS neurons through a sodium-dependent transporter named SVCT2 and participates in several CNS processes, for instance, the regulation of glutamate receptors functioning and the synthesis of neuromodulators. Here we studied the interplay between the adenosinergic system and ascorbate transport in neurons. We found that selective activation of A3, but not of A1 or A2a, adenosine receptors modulated ascorbate transport, decreasing intracellular ascorbate content. Förster resonance energy transfer (FRET) analyses showed that A3 receptors associate with the ascorbate transporter SVCT2, suggesting tight signaling compartmentalization between A3 receptors and SVCT2. The activation of A3 receptors increased ascorbate release in an SVCT2-dependent manner, which largely altered the neuronal redox status without interfering with cell death, glycolytic metabolism, and bioenergetics. Overall, by regulating vitamin C transport, the adenosinergic system (via activation of A3 receptors) can regulate ascorbate bioavailability and control the redox balance in neurons.

The specific adenosine A3 receptor (A3AR) agonist (CF101) has potential for inflammation and pain in various disease, such as arthritis, cancer and neuropathic pain, while the role of A3AR in post-traumatic OA and the underlying mechanism is largely unknown. CF101 was orally administrated in OA rats induced by anterior cruciate ligament transection (ACLT) surgery, and the rat primary chondrocytes were stimulated by hydrogen peroxide (H

The adenosine A

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.

Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Adenosine is a purine nucleoside involved in numerous physiological processes; however, the role of adenosine receptors in T. gondii-induced trophoblast cell function has not been investigated until now. The goal of the present study was to evaluate the intracellular signaling pathways regulated by adenosine receptors using a HTR-8/SVneo trophoblast cell model of T. gondii infection. HTR8/SVneo human extravillous trophoblast cells were infected with or without T. gondii and then evaluated for cell morphology, intracellular proliferation of the parasite, adenosine receptor expression, TNF-α production and mitogen-activated protein (MAP) kinase signaling pathways triggered by adenosine A3 receptor (A3AR). HTR8/SVneo cells infected with T. gondii exhibited an altered cytoskeletal changes, an increased infection rate and reduced viability in an infection time-dependent manner. T. gondii significantly promoted increased TNF-α production, A3AR protein levels and p38, ERK1/2 and JNK phosphorylation compared to those observed in uninfected control cells. Moreover, the inhibition of A3AR by A3AR siRNA transfection apparently suppressed the T. gondii infection-mediated upregulation of TNF-α, A3AR production and MAPK activation. In addition, T. gondii-promoted TNF-α secretion was dramatically attenuated by pretreatment with PD098059 or SP600125. These results indicate that A3AR-mediated activation of ERK1/2 and JNK positively regulates TNF-α secretion in T. gondii-infected HTR8/SVneo cells.

Treating chronic pain using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance and withdrawal which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a 2-fold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord (DH-SC). Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A3 adenosine receptor (A3AR) signaling. Supplementing adenosine signaling with selective A3AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A3AR in the spinal cord with an ADK inhibitor or A3AR agonist was associated with reduced DH-SC expression of the NOD-like receptor pyrin domain-containing 3 (NLRP3; 60-75%), cleaved caspase 1 (40-60%), interleukin (IL)-1β (76-80%) and tumor necrosis factor (TNF; 50-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased 2-fold. In mice, A3AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A3AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A3AR agonists may be useful adjunct to opioids to manage their unwanted effects.

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.

Various adenosine receptor nucleoside-like ligands were found to modulate ATP hydrolysis by the multidrug transporter ABCG2. Both ribose-containing and rigidified (N)-methanocarba nucleosides (C2-, N(6)- and 5’-modified), as well as adenines (C2-, N(6)-, and deaza modified), were included. 57 compounds out of 63 tested either stimulated (50) or inhibited (7) basal ATPase activity. Structure-activity analysis showed a separation of adenosine receptor and ABCG2 activities. The 7-deaza modification had favorable effects in both (N)-methanocarba nucleosides and adenines. Adenine 37c (MRS7608) and (N)-methanocarba 7-deaza-5’-ethyl ester 60 (MRS7343) were found to be potent stimulators of ABCG2 ATPase activity with EC(50) values of 13.2 ± 1.7 and 13.2 ± 2.2 nM, respectively. Both had affinity in the micromolar range for A(3) adenosine receptor and lacked the 5’-amide agonist-enabling group (37c was reported as a weak A(3) antagonist, K(i) 6.82 μM). Compound 60 significantly inhibited ABCG2 substrate transport (IC(50) 0.44 μM). Docking simulations predicted the interaction of 60 with 21 residues in the drug-binding pocket of ABCG2.

Activation of mineralocorticoid receptor antagonists (MRAs) is cardioprotective; however, this property is lost upon blockade or inactivation of adenosine (ADO) receptor A2b. In this study, we investigated whether the effects of MRAs are mediated by an interaction between cardioprotective ADO receptors A1 and A3. Spironolactone (SPI) or eplerenone (EPL) increased ADO levels in the plasma of treated animals compared to control animals. SPI or EPL increased the protein and activity levels of ecto-5'-nucleotidase (NT5E), an enzyme that synthesizes ADO, compared to control. The levels of ADO deaminase (ADA), which degrades ADO, were not affected by SPI or EPL; however, the activity of ADA was reduced in SPI-treated rats compared to control. Using an isolated cardiomyocyte model, we found inotropic and chronotropic effects, and increased calcium transient [Ca2+]i in cells treated with ADO receptor A1 or A3 antagonists compared to control groups. Upon co-treatment with MRAs, EPL and SPI fully and partially reverted the effects of receptor A1 or A3 antagonism, respectively. Collectively, MRAs in vivo lead to increased ADO bioavailability. In vitro, the rapid effects of SPI and EPL are mediated by an interaction between ADO receptors A1 and A3.

Inosine monophosphate (IMP) is the intracellular precursor for both adenosine monophosphate and guanosine monophosphate and thus plays a central role in intracellular purine metabolism. IMP can also serve as an extracellular signaling molecule, and can regulate diverse processes such as taste sensation, neutrophil function, and ischemia-reperfusion injury. How IMP regulates inflammation induced by bacterial products or bacteria is unknown. In this study, we demonstrate that IMP suppressed tumor necrosis factor (TNF)-α production and augmented IL-10 production in endotoxemic mice. IMP exerted its effects through metabolism to inosine, as IMP only suppressed TNF-α following its CD73-mediated degradation to inosine in lipopolysaccharide-activated macrophages. Studies with gene targeted mice and pharmacological antagonism indicated that A2A , A2B, and A3 adenosine receptors are not required for the inosine suppression of TNF-α production. The inosine suppression of TNF-α production did not require its metabolism to hypoxanthine through purine nucleoside phosphorylase or its uptake into cells through concentrative nucleoside transporters indicating a role for alternative metabolic/uptake pathways. Inosine augmented IL-β production by macrophages in which inflammasome was activated by lipopolysaccharide and ATP. In contrast to its effects in endotoxemia, IMP failed to affect the inflammatory response to abdominal sepsis and pneumonia. We conclude that extracellular IMP and inosine differentially regulate the inflammatory response.

Following our study of 4′-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A(3)AR affinity, we incorporated dopamine-related N(6) substituents in the full agonist 5′-methylamide series. N(6)-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed K(i) (nM) 0.563 at hA(3)AR (~20,000-fold selective) and 1.54 at mA(3)AR. 2-Alkyl ethers maintained A(3) affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and β-arrestin 2 (βarr2)-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 mA(3)AR 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 mA(3)AR 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 A(3)AR agonism in vivo.

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 (K(i) = 2.40 nM) as a potent human A(3) adenosine receptor (hA(3)AR) agonist, and subtle chemical modification induced a shift from antagonist to agonist. We elucidated this shift by developing new hA(3)AR 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 Thr94(3.36) and His272(7.43) 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.

The A(3) adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A(3)AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A(3)AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A(3)AR antagonist for all three species tested, albeit a little less selective for mouse and rat A(3)AR in comparison to the human A(3)AR. DPTN’s K(i) values at respective A(1), A(2A), A(2B), and A(3) receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A(3)ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A(3)AR antagonists in rats and mice, we also re-examined other commonly used and selective A(3)AR antagonists under the same experimental conditions. The K(i) values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A(3)ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [(125)I]I-AB-MECA binding to mouse and rat A(3)ARs, while potent human A(3)AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A(3)AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A(3)AR-related mechanism or function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11302-021-09823-5.

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-1β, 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.

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

Recently, the involvement of the nervous system in the pathology of allergic diseases has attracted increasing interest. However, the precise pathophysiological role of enteric neurons in food allergies has not been elucidated. We report the presence of functional high-affinity IgE receptors (FcεRIs) in enteric neurons. FcεRI immunoreactivities were observed in approximately 70% of cholinergic myenteric neurons from choline acetyltransferase-eGFP mice. Furthermore, stimulation by IgE-antigen elevated intracellular Ca2+ concentration in isolated myenteric neurons from normal mice, suggesting that FcεRIs are capable of activating myenteric neurons. Additionally, the morphological investigation revealed that the majority of mucosal mast cells were in close proximity to enteric nerve fibers in the colonic mucosa of food allergy mice. Next, using a newly developed coculture system of isolated myenteric neurons and mucosal-type bone-marrow-derived mast cells (mBMMCs) with a calcium imaging system, we demonstrated that the stimulation of isolated myenteric neurons by veratridine caused the activation of mBMMCs, which was suppressed by the adenosine A3 receptor antagonist MRE 3008F20. Moreover, the expression of the adenosine A3 receptor gene was detected in mBMMCs. Therefore, in conclusion, it is suggested that, through interaction with mucosal mast cells, IgE-antigen-activated myenteric neurons play a pathological role in further exacerbating the pathology of food allergy.

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 A

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A

Chemotherapy-induced neuropathic pain (CINP) in both sexes compromises many current chemotherapeutics and lacks an FDA-approved therapy. We recently identified the sphingosine-1-phosphate receptor subtype 1 (S1PR1) and A3 adenosine receptor subtype (A3AR) as novel targets for therapeutic intervention. Our work in male rodents using paclitaxel, oxaliplatin, and bortezomib showed robust inhibition of CINP with either S1PR1 antagonists or A3AR agonists. The S1PR1 functional antagonist FTY720 (Gilenya) is FDA-approved for treating multiple sclerosis, and selective A3AR agonists are in advanced clinical trials for cancer and inflammatory disorders, underscoring the need for their expedited trials in patients with CINP as chemotherapy adjuncts. Our findings reveal that S1PR1 antagonists and A3AR agonists mitigate paclitaxel and oxaliplatin CINP in female and male rodents, but failed to block or reverse bortezomib-induced neuropathic pain (BINP) in females. Although numerous mechanisms likely underlie these differences, we focused on receptor levels. We found that BINP in male rats, but not in female rats, was associated with increased expression of A3AR in the spinal cord dorsal horn, whereas S1PR1 levels were similar in both sexes. Thus, alternative mechanisms beyond receptor expression may account for sex differences in response to S1PR1 antagonists. Morphine and duloxetine, both clinical analgesics, reversed BINP in female mice, demonstrating that the lack of response is specific to S1PR1 and A3AR agents. Our findings suggest that A3AR- and S1PR1-based therapies are not viable approaches in preventing and treating BINP in females and should inform future clinical trials of these drugs as adjuncts to chemotherapy.

Despite the recent advances in the life sciences and the remarkable investment in drug discovery research, the success rate of small-molecule drug development remains low. Safety is the second most influential factor of drug attrition in clinical studies; thus, the selection of compounds with fewer toxicity concerns is crucial to increase the success rate of drug discovery. Compounds that promiscuously bind to multiple targets are likely to cause unexpected pharmacological activity that may lead to adverse effects. Therefore, avoiding such compounds during early research stages would contribute to identifying compounds with a higher chance of success in the clinic. To evaluate the interaction profile against a wide variety of targets, we constructed a small-scale promiscuity panel (PP) consisting of eight targets (ROCK1, PDE4D2, GR, PPARγ, 5-HT2B, adenosine A3, M1, and GABAA) that were selected from diverse gene families. The validity of this panel was confirmed by comparison with the promiscuity index evaluated from larger-scale panels. Analysis of data from the PP revealed that both lipophilicity and basicity are likely to increase promiscuity, while the molecular weight does not significantly contribute. Additionally, the promiscuity assessed using our PP correlated with the occurrence of both in vitro cytotoxicity and in vivo toxicity, suggesting that the PP is useful to identify compounds with fewer toxicity concerns. In summary, this small-scale and cost-effective PP can contribute to the identification of safer compounds that would lead to a reduction in drug attrition due to safety issues.

Methotrexate (MTX) reduces inflammation by increasing extracellular adenosine levels in rheumatoid arthritis (RA) patients. Adenosine acts via G-protein coupled receptors; ADORA1, ADORA2a, ADORA2b and ADORA3. We studied if baseline expression of whole blood adenosine receptors can predict response to MTX. RA patients [American College of Rheumatology/European-League-Against-Rheumatism (EULAR) 2010 criteria], Disease modifying anti-rheumatic drug (DMARD) naive with active disease [Disease Activity Score 28 (DAS28) > 3.2] were enrolled. Blood samples were collected at baseline (n = 100) and at 4 months after therapy (n = 50). Patients were treated with MTX monotherapy. Based on EULAR response, patients were categorized into three groups i.e. good, moderate and non-responders. Adenosine receptors gene expression (ADORA1, ADORA2a, ADORA2b and ADORA3) in whole-blood RNA was measured using real-time PCR. HPRT1 was used as housekeeping gene. Receptor expression at baseline was correlated with response to MTX. All values are expressed as median (interquartile range). Hundred patients [87% females; age 40 (18) years]; duration of disease 24 (24.75) months; DAS28 4.7 (1.25) were enrolled. Fifty-one were classified as good, 28 moderate and 21 as non-responders. No expression of ADORA1 and ADORA2b was detected. Significant difference was observed in the expression levels of ADORA3 between good vs non-responder (P = 0.03) and moderate vs non-responder (P = 0.002). On ROC curve analysis, ADORA3 with cut-off value of less than − 0.60 (ΔCt) predicted non-response to MTX treatment (AUC: 0.7, P = 0.006). ADORA3 mRNA levels in whole blood may serve as a biomarker of response to MTX.

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A3 receptor (A3AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A3AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A3AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A3AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A3AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([3H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [3H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.

Background The pain-relief properties of tricyclic antidepressants can be attributed to several actions. Recent observations suggest that adenosine is involved in the antinociceptive effect of amitriptyline. The A3 adenosine receptor (A3AR) is the only adenosine subtype overexpressed in inflammatory and cancer cells. This study was performed to investigate the role of A3AR in the anti-nociceptive effect of amitriptyline. Methods Spinal nerve-ligated neuropathic pain was induced by ligating the L5 and L6 spinal nerves of male Sprague-Dawley rats. The neuropathic rats were randomly assigned to one of the following three groups (8 per group): a neuropathic pain with normal saline group, a neuropathic pain with amitriptyline group, and a neuropathic pain with amitriptyline and 3-ethyl-5-benzyl- 2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS) group. Amitriptyline or saline was administered intraperitoneally and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191), an A3AR antagonist, was injected subcutaneously immediately before amitriptyline administration. The level of extracellular signal-regulated kinase P44/42 (ERK1/2), cyclic AMP response element-binding protein (CREB), and proinflammatory cytokines were assessed using immunoblotting or reverse-transciption polymerase chain reaction. Results Amitriptyline increased the mechanical withdrawal threshold of the neuropathic rats. The level of phospho-ERK1/2 and phospho-CREB proteins, and proinflammatory cytokines produced by spinal nerve ligation were significantly reduced by amitriptyline administration. However, the use of MRS-1191 before amitriptyline administration not only reduced the threshold of mechanical allodynia, but also increased the signaling protein and proinflammatory cytokine levels, which were reduced by amitriptyline. Conclusions The results of this study suggest that the anti-nociceptive effect of amitriptyline involves the suppression of ERK1/2 and CREB signaling proteins, and A3AR activation also affects the alleviation of the inflammatory response.

Cancer patients undergoing immunotherapy may develop cytokine release syndrome (CRS), an inflammatory cytokine storm condition, followed by neurotoxic manifestations and may be life-threatening. The current treatments for CRS successfully reduce the inflammatory response but may limit the anticancer effect of the given immunotherapy and fail to overcome the neurotoxic adverse events. Adenosine, a ubiquitous purine nucleoside, induces a plethora of effects in the body via its binding to four adenosine receptors A1, A2a, A2b, and the A3. Highly selective agonists to the A3 adenosine receptor act as inhibitors of proinflammatory cytokines, possess robust anti-inflammatory and anticancer activity, and concomitantly, induce neuroprotective effects. Piclidenoson and namodenoson belong to this group of compounds, are effective upon oral administration, show an excellent safety profile in human clinical studies, and therefore, may be considered as drug candidates to treat CRS. In this article, the detailed anti-inflammatory characteristics of these compounds and the rationale to use them as drugs to combat CRS are described.

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.

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.

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 G(i) protein-coupled A(3) receptor (A(3)R) for adenosine has been suggested as a novel and very promising therapeutic target for psoriasis. Accordingly, selective, and high affinity A(3)R agonists are known to induce robust anti-inflammatory effects in animal models of autoimmune inflammatory diseases. Here, we demonstrated the efficacy of a selective A(3)R 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 A(3)R 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.

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.

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-1β, 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.

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 (A155 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.Expression of ALJ529 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 is a natural nucleoside present in a variety of organs and tissues, where it acts as a modulator of diverse physiological and pathophysiological processes. These actions are mediated by at least four G protein-coupled receptors, which are widely and differentially expressed in tissues. Interestingly, high concentrations of adenosine have been reported in a variety of tumors. In this context, the final output of adenosine in tumorigenesis will likely depend on the constellation of adenosine receptors expressed by tumor and stromal cells. Notably, activation of the A3 receptor can reduce the proliferative capacity of various cancer cells.This study aimed to describe the anti-proliferative effects of two previously synthesized adenosine derivatives with A3 agonist action (compounds 2b and 2f) through in vitro assays.We used gastric and breast cancer cell lines expressing the A3 receptor as in vitro models and theoretical experiments for molecular dynamics and determination of ADME properties.The antiproliferative effects of adenosine derivatives (after determining IC50 values) were comparable or even higher than those described for IB-MECA, a commercially available A3 agonist. Among possible mechanisms involved, apoptosis was found to be induced in MCF-7 cells but not in AGS or MDA-MB-231 cells. Surprisingly, we were unable to observe cellular senescence induction upon treatment with compounds 2b and 2f in any of the cell lines studied, although we cannot rule out other forms of cell cycles exit at this point.Both adenosine derivatives showed antiproliferative effects on gastric and breast cancer cell lines, and were able to induce apoptosis, at least in the MCF-7 cell line. Further studies will be necessary to unveil receptor specificity and mechanisms accounting for the antiproliferative properties of these novel semi-synthetic compounds.

Novel coronavirus disease 2019 (COVID-19) causes pulmonary and cardiovascular disorders and has become a worldwide emergency. Myocardial injury can be caused by direct or indirect damage, particularly mediated by a cytokine storm, a disordered immune response that can cause myocarditis, abnormal coagulation, arrhythmia, acute coronary syndrome, and myocardial infarction. The present review focuses on the mechanisms of this viral infection, cardiac biomarkers, consequences, and the possible therapeutic role of purinergic and adenosinergic signalling systems. In particular, we focus on the interaction of the extracellular nucleotide adenosine triphosphate (ATP) with its receptors P2X1, P2X4, P2X7, P2Y1, and P2Y2 and of adenosine (Ado) with A2A and A3 receptors, as well as their roles in host immune responses. We suggest that receptors of purinergic signalling could be ideal candidates for pharmacological targeting to protect against myocardial injury caused by a cytokine storm in COVID-19, in order to reduce systemic inflammatory damage to cells and tissues, preventing the progression of the disease by modulating the immune response and improving patient quality of life.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

Background Traumatic brain injury (TBI) is a common pathological condition that presently lacks a specific pharmacological treatment. Adenosine levels rise following TBI, which is thought to be neuroprotective against secondary brain injury. Evidence from stroke and inflammatory disease models suggests that adenosine signaling through the G protein-coupled A3 adenosine receptor (A3AR) can provide antiinflammatory and neuroprotective effects. However, the role of A3AR in TBI has not been investigated. Methods Using the selective A3AR agonist, MRS5980, we evaluated the effects of A3AR activation on the pathological outcomes and cognitive function in CD1 male mouse models of TBI. Results When measured 24 h after controlled cortical impact (CCI) TBI, male mice treated with intraperitoneal injections of MRS5980 (1 mg/kg) had reduced secondary tissue injury and brain infarction than vehicle-treated mice with TBI. These effects were associated with attenuated neuroinflammation marked by reduced activation of nuclear factor of kappa light polypeptide gene enhancer in B cells (NFκB) and MAPK (p38 and extracellular signal-regulated kinase (ERK)) pathways and downstream NOD-like receptor pyrin domain-containing 3 inflammasome activation. MRS5980 also attenuated TBI-induced CD4+ and CD8+ T cell influx. Moreover, when measured 4–5 weeks after closed head weight-drop TBI, male mice treated with MRS5980 (1 mg/kg) performed significantly better in novel object-placement retention tests (NOPRT) and T maze trials than untreated mice with TBI without altered locomotor activity or increased anxiety. Conclusion Our results provide support for the beneficial effects of small molecule A3AR agonists to mitigate secondary tissue injury and cognitive impairment following TBI.

IL-6-triggered Th17 cell expansion is responsible for the pathogenesis of many immune diseases including rheumatoid arthritis (RA). Traditionally, IL-6 induces Th17 cell differentiation through JAK-STAT3 signaling. In the present work, PKA inhibition reduces in vitro induction of Th17 cells, while IL-6 stimulation of T cells facilitates the internalization of A

Macrophages perform the fundamental function of sensing cellular damage, initiating and mediating immune response and tissue repair. Adenine nucleotides are in relatively high abundance in cells and are released from cells during tissue damage that are converted to adenosine in the extracellular environment. The A1, A2A, A2B and A3 adenosine receptors serve to regulate immune function. Despite characterization of the adenosine receptors, a comprehensive examination of adenosine receptor signaling in THP-1 macrophage cells has not been done. Moreover, previous studies employed chemical agonists and antagonists that have the potential for off-target affects. Here we systematically knockdown each of the four known adenosine receptors in THP-1 macrophages using validated siRNA and investigated their function under LPS stimulation. We demonstrate that the A1 receptor is required for adenosine-stimulated IL-10 and IL-1β secretion indicating an important role of this receptor during resolution of inflammation and tissue repair in these cells. The A1 and A3 receptor were required for IL-6 and IL-1β secretion showing a net pro-inflammatory role for these receptors. Finally, we present the novel finding that THP-1 macrophages lacking the A2B receptor undergo pyroptosis when exposed to LPS, demonstrating a novel role of the A2B receptor in regulation of programmed cell death during inflammation. This work underscores the fundamental importance of adenosine signaling and provides insight into the independent roles of the adenosine receptors in modulating cytokine signaling.

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 A

The A

The adenosine A3 receptor (A3R) is the only adenosine receptor subtype to be overexpressed in inflammatory and cancer cells and therefore is considered a novel and promising therapeutic target for inflammatory diseases and cancer. Heterologous expression of A3R at levels to allow biophysical characterization is a major bottleneck in structure-guided drug discovery efforts. Here, we apply protein engineering using chimeric receptors to improve expression and activity in yeast. Previously we had reported improved expression and trafficking of the chimeric A1R variant using a similar approach. In this report, we constructed chimeric A3/A2AR comprising the N-terminus and transmembrane domains from A3R (residues 1&ndash, 284) and the cytoplasmic C-terminus of the A2AR (residues 291&ndash, 412). The chimeric receptor showed approximately 2-fold improved expression with a 2-fold decreased unfolded protein response when compared to wild type A3R. Moreover, by varying culture conditions such as initial cell density and induction temperature a further 1.7-fold increase in total receptor yields was obtained. We observed native-like coupling of the chimeric receptor to Gai-Gpa1 in engineered yeast strains, activating the downstream, modified MAPK pathway. This strategy of utilizing chimeric receptor variants in yeast thus provides an exciting opportunity to improve expression and activity of &ldquo, difficult-to-express&rdquo, receptors, expanding the opportunity for utilizing yeast in drug discovery.

The aim of this paper was to check the possible interaction of two of the four purinergic P1 receptors, the A2A and the A3. Discovery of the A2A–A3 receptor complex was achieved by means of immunocytochemistry and of bioluminescence resonance energy transfer. The functional properties and heteromer print identification were addressed by combining binding and signaling assays. The physiological role of the novel heteromer is to provide a differential signaling depending on the pre-coupling to signal transduction components and/or on the concentration of the endogenous agonist. The main feature was that the heteromeric context led to a marked decrease of the signaling originating at A3 receptors. Interestingly from a therapeutic point of view, A2A receptor antagonists overrode the blockade, thus allowing A3 receptor-mediated signaling. The A2A–A3 receptor heteromer print was detected in primary cortical neurons. These and previous results suggest that all four adenosine receptors may interact with each other. Therefore, each adenosine receptor could form heteromers with distinct properties, expanding the signaling outputs derived from the binding of adenosine to its cognate receptors.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure–activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics—Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (

Methotrexate (MTX) administered at the dose 10-15 mg/mOne hundred children with JIA of all subtypes treated with MTX were recruited to the study. Demographic and clinical parameters were collected at the baseline of MTX therapy and on a control visit 4-6 months after starting MTX. Single nucleotide polymorphism genotyping was performed using genomic DNA isolated from peripheral blood samples.The polymorphic variant of ADORA2A rs2236624 was associated with ~3.5 times higher odds of gastrointestinal side effects occurrence (odds ratio: 3.59, 95% CI: 1.15-11.22, P = 0.0282). Children with the ADORA3 rs3393 polymorphic variants (CT/CC) after 6 months of MTX treatment had significantly lower number of joints with active arthritis (median: 0.00 vs 1.00, P = 0.0400) and value of C-reactive protein (0.60 vs 2.40, P = 0.0242) in comparison to TT variant.Although future studies are needed to verify our findings, polymorphisms in ADORA2A and ADORA3 genes may become the determinants of MTX treatment efficacy and gastrointestinal toxicity in children with JIA.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y(1) receptor (P2Y(1)R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A(3) and A(1)ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y(1)R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

Dopamine-derived N(6)-substituents, compared to N(6)-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A(3) adenosine receptor (AR) affinity/selectivity, e.g. C2-phenylethynyl analogue 15 (MRS7591, K(i) 10.9/17.8 nM, at human/mouse A(3)AR). 15 was a partial agonist in vitro (hA(3)AR, cAMP inhibition, 31% E(max); mA(3)AR, [(35)S]GTP-γ-S binding, 16% E(max)) and in vivo, and also antagonized hA(3)AR in vitro. Distal H-bonding substitutions of the N(6)-(2-phenylethyl) moiety particularly enhanced mA(3)AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA(3)AR and hA(3)AR homology models. These hybrid models were based on an inactive antagonist-bound hA(1)AR structure for the upper part of TM2 and an agonist-bound hA(2A)AR structure for the remaining TM portions. These species-independent A(3)AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A(3)AR, a drug target of growing interest.

The A(3) adenosine receptor (A(3)AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable β-arrestin 2 (βarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGα(i) protein to activated A(3)AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N(6)-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the βarr2 or miniGα(i) pathway.