Optochemical control of slow-wave sleep in the nucleus accumbens of male mice by a photoactivatable allosteric modulator of adenosine A2A receptors.

Optochemistry, an emerging pharmacologic approach in which light is used to selectively activate or deactivate molecules, has the potential to alleviate symptoms, cure diseases, and improve quality of life while preventing uncontrolled drug effects. The development of in-vivo applications for optochemistry to render brain cells photoresponsive without relying on genetic engineering has been progressing slowly. The nucleus accumbens (NAc) is a region for the regulation of slow-wave sleep (SWS) through the integration of motivational stimuli. Adenosine emerges as a promising candidate molecule for activating indirect pathway neurons of the NAc expressing adenosine A2A receptors (A2ARs) to induce SWS. Here, we developed a brain-permeable positive allosteric modulator of A2ARs (A2AR PAM) that can be rapidly photoactivated with visible light (λ > 400 nm) and used it optoallosterically to induce SWS in the NAc of freely behaving male mice by increasing the activity of extracellular adenosine derived from astrocytic and neuronal activity.

Membrane mimetic-dependence of GPCR energy landscapes.

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

Population Pharmacokinetics of the Novel Adenosine A2A Antagonist/Inverse Agonist KW-6356 and Its Active Metabolite Following Single and Multiple Oral Administration in Healthy Individuals and Patients with Parkinson's Disease.

KW-6356 is a selective antagonist and inverse agonist of the adenosine A2A receptor. The primary aim of the present analysis was to characterize the pharmacokinetics (PK) of KW-6356 and its active metabolite M6 in healthy subjects and patients with Parkinson's disease (PD). We pooled concentration-time data from healthy subjects and patients with PD who were administered KW-6356. Using these data, we developed a population PK model by sequentially fitting the KW-6356 parameters followed by the M6 parameters. A first-order absorption with a 1-compartment model for KW-6356 and a 1-compartment model for M6 best described the profiles. The covariates included in the final models were food status (fed/fasted/unknown) on first-order absorption rate constant, baseline serum albumin level on apparent clearance of KW-6356, and baseline body weight on apparent volume of distribution of KW-6356 and apparent clearance of M6. No covariate had a clinically meaningful impact on KW-6356 or M6 exposure.

A2aR inhibits fibrosis and the EMT process in silicosis by regulating Wnt/ß-catenin pathway.

Silicosis, a disease characterized by diffuse fibrosis of the lung tissue, is caused by long-term inhalation of free silica (SiO2) dust in the occupational environment and is currently the most serious occupational diseases of pneumoconiosis. Several studies have suggested that alveolar type Ⅱ epithelial cells (AEC Ⅱ) undergo epithelial-mesenchymal transition (EMT) as one of the crucial components of silicosis in lung fibroblasts. A2aR can play a critical regulatory role in fibrosis-related diseases by modulating the Wnt/ß-catenin pathway, but its function in the EMT process of silicosis has not been explained. In this study, an EMT model of A549 cells was established. The results revealed that A2aR expression is reduced in the EMT model. Furthermore, activation of A2aR or suppression of the Wnt/ß-catenin pathway reversed the EMT process, while the opposite result was obtained by inhibiting A2aR. In addition, activation of A2aR in a mouse silicosis model inhibited the Wnt/ß-catenin pathway and ameliorated the extent of silica-induced lung fibrosis in mice. To sum up, we uncovered that A2aR inhibits fibrosis and the EMT process in silicosis by regulating the Wnt/ß-catenin pathway. Our study can provide an experimental basis for elucidating the role of A2aR in the development of silicosis and offer new ideas for further exploration of interventions for silicosis.

Role of adenosine A2A receptors in the loss of consciousness induced by propofol anesthesia.

The mechanism of propofol-anesthesia-induced loss of consciousness (LOC) remains largely unknown. We speculated that the adenosine A2A receptor serves as a vital molecular target in regulating LOC states under propofol anesthesia. c-Fos staining helped observe the changes in the neuronal activity in the nucleus accumbens (NAc). Initially, the adenosine signals in the NAc were measured under propofol anesthesia using fiber photometry recordings. Then, behavior tests and electrophysiological recordings were used to verify the effect of systemic A2A R agonist or antagonist treatment on propofol anesthesia. Next, the microinjection technique was used to clarify the role of the NAc A2A R under propofol anesthesia. Fiber photometry recordings were applied to assess the effect of A2A R agonist or antagonist systemic treatment on adenosine signal alterations in the NAc during propofol anesthesia. Then, as the GABAergic neurons are the main neurons in the NAc, we further measured the neuronal activity of GABAergic neurons. In our study, propofol anesthesia enhanced the neuronal activity in the NAc, and the adenosine signals were increased in the NAc. SCH58261 reduced the LOC time and sedative depth, while CGS21680 increased those via intraperitoneal injection. Additionally, CGS21680 increased the changes in delta, theta, alpha, beta, and low-gamma oscillations in the NAc. Moreover, microinjection of SCH58261 significantly shortened the LOC time, whereas microinjection of CGS21680 into the NAc significantly prolonged the LOC duration. The results illustrated that after A2A R agonist administration, the level of extracellular adenosine signals in the NAc was decreased and the neuronal activity of GABAergic neurons was enhanced, whereas after A2A R antagonist administration via intraperitoneal injection, the opposite occurred. This study reveals the vital role of the A2A R in propofol-induced LOC and that the A2A R could affect the maintenance of propofol anesthesia.

[Activation of the adenosine A2A receptor at the acute stage of moderate traumatic brain injury enhances the neuroprotective effects of oxaloacetate].

The purpose of the present study was to investigate the effect of glutamate scavenger oxaloacetate (OA) combined with CGS21680, an adenosine A2A receptor (A2AR) agonist, on acute traumatic brain injury (TBI), and to elucidate the underlying mechanisms. C57BL/6J mice were subjected to moderate-level TBI by controlled cortical impact, and then were treated with OA, CGS21680, or OA combined with CGS21680 at acute stage of TBI. At 24 h post TBI, neurological severity score, brain water content, glutamate concentration in cerebrospinal fluid (CSF), mRNA and protein levels of IL-1ß and TNF-α, mRNA level and activity of glutamate oxaloacetate aminotransferase (GOT), and ATP level of brain tissue were detected. The results showed that neurological deficit, brain water content, glutamate concentration in CSF, and the inflammatory cytokine IL-1ß and TNF-α production were exacerbated in CGS21680 treated mice. Administrating OA suppressed the rise of both glutamate concentration in CSF and brain water content, and elevated the ATP level of cerebral tissue. More interestingly, neurological deficit, brain edema, glutamate concentration, IL-1ß and TNF-α levels were ameliorated significantly in mice treated with OA combined with CGS21680. The combined treatment exhibited better therapeutic effects than single OA treatment. We also observed that GOT activity was enhanced in single CGS21680 treatment group, and both the GOT mRNA level and GOT activity were up-regulated in early-stage combined treatment group. These results suggest that A2AR can improve the efficiency of GOT and potentiate the ability of OA to metabolize glutamate. This may be the mechanism that A2AR activation in combination group augmented the neuroprotective effect of OA rather than aggravated the brain damages. Taken together, the present study provides a new insight for the clinical treatment of TBI with A2AR agonists and OA.

Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif.

BACKGROUND: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. METHODS: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. RESULTS AND CONCLUSIONS: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Regionally selective cardiovascular responses to adenosine A2A and A2B receptor activation.

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein-coupled receptors (A1 , A2A , A2B , A3 ). Here, we have investigated the effect of A2A and A2B -selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A2A -selective agonist CGS 21680 (0.1, 0.3, 1 µg kg-1 min-1 ) or the A2B -selective agonist BAY 60-6583 (4,13.3, 40 µg kg-1 min-1 ) following predosing with the A2A -selective antagonist SCH 58261 (0.1 or 1 mg kg-1 min-1 ), the A2B /A2A antagonist PSB 1115 (10 mg kg-1 min-1 ) or vehicle. The A2A -selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B -selective agonist BAY 60-6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury.

Allosteric Modulation of Adenosine A2A Receptors as a New Therapeutic Avenue.

The therapeutic potential of targeting adenosine A2A receptors (A2ARs) is immense due to their broad expression in the body and central nervous system. The role of A2ARs in cardiovascular function, inflammation, sleep/wake behaviors, cognition, and other primary nervous system functions has been extensively studied. Numerous A2AR agonist and antagonist molecules are reported, many of which are currently in clinical trials or have already been approved for treatment. Allosteric modulators can selectively elicit a physiologic response only where and when the orthosteric ligand is released, which reduces the risk of an adverse effect resulting from A2AR activation. Thus, these allosteric modulators have a potential therapeutic advantage over classical agonist and antagonist molecules. This review focuses on the recent developments regarding allosteric A2AR modulation, which is a promising area for future pharmaceutical research because the list of existing allosteric A2AR modulators and their physiologic effects is still short.

Adenosine 2A receptor and TIM3 suppress cytolytic killing of tumor cells via cytoskeletal polarization.

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.

Secondary Burn Progression Mitigated by an Adenosine 2A Receptor Agonist.

Current burn therapy is largely supportive with limited therapies to curb secondary burn progression. Adenosine 2A receptor (A2AR) agonists have anti-inflammatory effects with decreased inflammatory cell infiltrate and release of proinflammatory mediators. Using a porcine comb burn model, we examined whether A2AR agonists could mitigate burn progression. Eight full-thickness comb burns (four prongs with three spaces per comb) per pig were generated with the following specifications: temperature 115°C, 3-kg force, and 30-second application time. In a randomized fashion, animals (four per group) were then treated with A2AR agonist (ATL-1223, 3 ng/kg/min, intravenous infusion over 6 hours) or vehicle control. Necrotic interspace development was the primary outcome and additional histologic assessments were conducted. Analysis of unburned interspaces (72 per group) revealed that ATL-1223 treatment decreased the rate of necrotic interspace development over the first 4 days following injury (p < .05). Treatment significantly decreased dermal neutrophil infiltration at 48 hours following burn (14.63 ± 4.30 vs 29.71 ± 10.76 neutrophils/high-power field, p = .029). Additionally, ATL-1223 treatment was associated with fewer interspaces with evidence of microvascular thrombi through postburn day 4 (18.8% vs 56.3%, p = .002). Two weeks following insult, the depth of injury at distinct burn sites (adjacent to interspaces) was significantly reduced by ATL-1223 treatment (2.91 ± 0.47 vs 3.28 ± 0.58 mm, p = .038). This work demonstrates the ability of an A2AR agonist to mitigate burn progression through dampening local inflammatory processes. Extended dosing strategies may yield additional benefit and improve cosmetic outcome in those with severe injury.

Effect of S-Se Bioisosteric Exchange on Affinity and Intrinsic Efficacy of Novel N-acylhydrazone Derivatives at the Adenosine A2A Receptor.

In this work, we evaluated the conformational effect promoted by the isosteric exchange of sulfur by selenium in the heteroaromatic ring of new N-acylhydrazone (NAH) derivatives (3-8, 13, 14), analogues of the cardioactive compounds LASSBio-294 (1) and LASSBio-785 (2). NMR spectra analysis demonstrated a chemical shift variation of the iminic Csp2 of NAH S/Se-isosters, suggesting a stronger intramolecular chalcogen interaction for Se-derivatives. To investigate the pharmacological profile of these compounds at the adenosine A2A receptor (A2AR), we performed a previously validated functional binding assay. As expected for bioisosteres, the isosteric-S/Se replacement affected neither the affinity nor the intrinsic efficacy of our NAH derivatives (1-8). However, the N-methylated compounds (2, 6-8) presented a weak partial agonist profile at A2AR, contrary to the non-methylated counterparts (1, 3-5), which appeared as weak inverse agonists. Additionally, retroisosterism between aromatic rings of NAH on S/Se-isosters mimicked the effect of the N-methylation on intrinsic efficacy at A2AR, while meta-substitution in the phenyl ring of the acyl moiety did not. This study showed that the conformational effect of NAH-N-methylation and aromatic rings retroisosterism changed the intrinsic efficacy on A2AR, indicating the S/Se-chalcogen effect to drive the conformational behavior of this series of NAH.

Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation.

Hepatic ischemia/reperfusion injury (IRI) is aggravated by steatosis and is a main risk factor in fatty liver transplantation. Adenosine receptors (ARs) are emerging as therapeutic targets in liver diseases. By using cellular and in vivo systems of hepatic steatosis and IRI, here we evaluated the effects of pharmacological A2AR and A1R activation. The A2AR agonist CGS21680 protected the primary steatotic murine hepatocyte from IR damage and the activation of ASK1 and JNK. Such an effect was attributed to a phosphatidylinositol-3-kinase (PI3K)/Akt-dependent inhibition of ASK1. By contrast, the A1R agonist CCPA enhanced IR damage, intracellular steatosis and oxidative species (OS) production, thereby further increasing the lipid/OS-dependent ASK1-JNK stimulation. The CGS2680 and CCPA effects were nullified by a genetic ASK1 downregulation in steatotic hepatoma C1C7 cells. In steatotic mice livers, CGS21680 protected against hepatic IRI and ASK1/JNK activation whereas CCPA aggravated hepatic steatosis and IRI, and enhanced ASK1 and JNK stimulation. These results evidence a novel mechanism of CGS21680-mediated hepatoprotection, i.e., the PI3K/AKT-dependent inhibition of ASK1, and they show that CGS21680 and CCPA reduces and enhances the IRI of fatty liver, respectively, by preventing or increasing the activation of the cytotoxic ASK1/JNK axis. They also indicate the selective employment of A2AR agonists as an effective therapeutic strategy to prevent IRI in human fatty liver surgery.

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations.

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.

Structure-Based Design of Dual-Acting Compounds Targeting Adenosine A2A Receptor and Histone Deacetylase as Novel Tumor Immunotherapeutic Agents.

Adenosine is an immunosuppressive factor in the tumor microenvironment mainly through activation of the A2A adenosine receptor (A2AR), which is a mechanism hijacked by tumors to escape immune surveillance. Small-molecule A2AR antagonists are being evaluated in clinical trials as immunotherapeutic agents, but their efficacy is limited as standalone therapies. To enhance the antitumor effects of A2AR antagonists, dual-acting compounds incorporating A2AR antagonism and histone deacetylase (HDAC) inhibitory actions were designed and synthesized, based on co-crystal structures of A2AR. Compound 24e (IHCH-3064) exhibited potent binding to A2AR (Ki = 2.2 nM) and selective inhibition of HDAC1 (IC50 = 80.2 nM), with good antiproliferative activity against tumor cell lines in vitro. Intraperitoneal administration of 24e (60 mg/kg, bid) inhibited mouse MC38 tumor growth with a tumor growth inhibition rate of 95.3%. These results showed that dual-acting compounds targeting A2AR and HDAC are potentially immunotherapeutic agents that are worth further exploring.

Myelin Defects in Niemann-Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives.

Niemann-Pick type C (NPC) disease is a wide-spectrum clinical condition classified as a neurovisceral disorder affecting mainly the liver and the brain. It is caused by mutations in one of two genes, NPC1 and NPC2, coding for proteins located in the lysosomes. NPC proteins are deputed to transport cholesterol within lysosomes or between late endosome/lysosome systems and other cellular compartments, such as the endoplasmic reticulum and plasma membrane. The first trait of NPC is the accumulation of unesterified cholesterol and other lipids, like sphingosine and glycosphingolipids, in the late endosomal and lysosomal compartments, which causes the blockade of autophagic flux and the impairment of mitochondrial functions. In the brain, the main consequences of NPC are cerebellar neurodegeneration, neuroinflammation, and myelin defects. This review will focus on myelin defects and the pivotal importance of cholesterol for myelination and will offer an overview of the molecular targets and the pharmacological strategies so far proposed, or an object of clinical trials for NPC. Finally, it will summarize recent data on a new and promising pharmacological perspective involving A2A adenosine receptor stimulation in genetic and pharmacological NPC dysmyelination models.

Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats.

Neuroplasticity is a fundamental property of the respiratory control system, enabling critical adaptations in breathing to meet the challenges, but little is known whether neonates express neuroplasticity similar to adults. We tested the hypothesis that, similar to adults, tyrosine receptor kinase B (TrkB) or adenosine A2a receptor activation in neonates are independently sufficient to elicit respiratory motor facilitation, and that co-induction of TrkB and A2a receptor-dependent plasticity undermines respiratory motor facilitation. TrkB receptor activation with 7,8-dihydroxyflavone (DHF) in neonatal brainstem-spinal cord preparations induced a long-lasting increase in respiratory motor output in 55 % of preparations, whereas adenosine A2a receptor activation with CGS21680 only sporadically induced respiratory motor plasticity. CGS21680 and DHF co-application prevented DHF-dependent respiratory motor facilitation, whereas co-application of MSX-3 (adenosine A2a receptor antagonist) and DHF more rapidly induced respiratory motor plasticity. Collectively, these data suggest that mechanisms underlying respiratory neuroplasticity may be only partially operational in early neonatal life, and that adenosine A2a receptor activation undermines TrkB-induced respiratory plasticity.

A2A receptor agonists and P2Y12 receptor antagonists modulate expression of thrombospondin-1 (TSP-1) and its secretion from Human Microvascular Endothelial Cells (HMEC-1).

BACKGROUNDS AND AIMS: To address the problem of resistance to standard antiplatelet therapy in some patients, our team proposed a purinoceptor-dependent dual therapy. Its efficacy is also determined by the condition of the vascular endothelium which, by secreting numerous factors, is involved in hemostasis. Among them, thrombospondin-1 is important in the context of thrombotic events. Therefore we sought to determine if the novel dual purinoceptor-dependent concept is associated with TSP-1 changes in vascular endothelial cells. METHODS AND RESULTS: TSP-1 expression in human microvascular endothelial cells was determined at transcriptional and protein level. We performed real-time PCR, the Western blot analysis and ELISA test. We found that TSP-1 mRNA and protein expression levels significantly changed in response to P1R agonists treatment. Furthermore, we have observed that co-administration of selective A2AR agonists (UK-432,097 or MRE0094) with P2Y12R antagonists altered TSP-1 expression levels, and the direction of these changes was not synergistic. MRE0094 applied with ARC69931MX or R-138727 increased mRNA expression from 39 to 56 or 57%, respectively (*P < 0.05 vs. MRE0094; ***P < 0.001 vs. control). Also, in the case of the P2Y12R antagonists used together with UK-432,097, there was an increase from 53 to 71 and 70% (*P < 0.05 vs. UK-432,097; ***P < 0.001 vs. control). The observed trends in gene expression were reflected in the protein expression and the level of its secretion from HMEC-1. CONCLUSION: The article presents evidence which proves that the purinoceptor-dependent concept is associated with TSP-1 changes in endothelial cells (EC). Moreover, Human Microvascular Endothelial Cells treatment applied together with agonists (MRE0094 or UK-432,097) and P2Y12R antagonist did not result in any synergistic effect, implicating a possible crosstalk between G proteins in GPCRs dependent signaling. Therefore, we suggest that understanding of the specific mechanism underlying TSP-1 alterations in EC in the context of the dual purinoceptor-dependent approach is essential for antiplatelet therapies and should be the subject of future research.

Affinity Mass Spectrometry-Based Fragment Screening Identified a New Negative Allosteric Modulator of the Adenosine A2A Receptor Targeting the Sodium Ion Pocket.

Allosteric ligands provide new opportunities to modulate G protein-coupled receptor (GPCR) function and present therapeutic benefits over orthosteric molecules. Negative allosteric modulators (NAMs) can inhibit the activation of a receptor and downstream signal transduction. Screening NAMs for a GPCR target is particularly challenging because of the difficulty in distinguishing NAMs from antagonists bound to the orthosteric site as they both show inhibitory effects in receptor signaling assays. Here we report an affinity mass spectrometry (MS)-based approach tailored to screening potential NAMs of a GPCR target especially from fragment libraries. Compared to regular surface plasmon resonance or NMR-based methods for fragment screening, our approach features a reduction of the protein and compound consumption by 2-4 orders of magnitude and an increase in the data acquisition speed by 2-3 orders of magnitude. Our affinity MS-based fragment screening led to the identification of a new NAM of the adenosine A2A receptor (A2AAR) bearing an unprecedented azetidine moiety predicted to occupy the allosteric sodium binding site. Molecular dynamics simulations, ligand structure-activity relationship (SAR) studies, and in-solution NMR analyses further revealed the unique binding mode and antagonistic property of this compound that differs considerably from HMA (5-(N,N-hexamethylene)amiloride), a well-characterized NAM of A2AAR. Taken together, our work would facilitate fragment-based screening of allosteric modulators, as well as guide the design of novel NAMs acting at the sodium ion pocket of class A GPCRs.

The role of peripheral adenosine receptors in glutamate-induced pain nociceptive behavior.

The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 µmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 µg/paw significantly reduced glutamate-induced nociception (p<0.01 and p<0.001 vs. vehicle, respectively, n=8-10). In contrast, i.pl. injection of hydrochloride hydrate (CGS21680, an adenosine A2A receptor agonist) (1 µg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 µg/paw) caused a significant reduction (p<0.05, n=7-8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs-8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1-10 µg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1-100 µg/paw), alloxazine (an A2B antagonist, 0.1-3 µg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1-100 µg/paw) (p>0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 µg/paw) significantly blocked the anti-nociceptive effect of CHA (1 µg/paw) (p<0.05, n=7-9). Similarly, ZM241385 (20 µg/paw) administered prior to CGS21680 (1 µg/paw) significantly blocked CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 µg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7-8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 µg/paw) (p<0.05, n=7-9) but not by the A2A antagonist ZM241385 (10 µg/paw, p>0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.