Adenosine A1 Receptor Research Articles

Hypnotic effect of AR-001 through adenosine A1 receptor

Insomnia is one of the most common sleep disorders, affecting 10–15% of the global population. Because classical remedies used to treat insomnia have various side effects, new therapeutics for insomnia are attracting attention. In the present study, we found that N2-Ethyl-N4-(furan-2-ylmethyl) quinazoline-2,4-diamine (AR-001) has adenosine A1 receptor agonistic activity and exhibits hypnotic efficacy by decreasing sleep onset latency and increasing total sleep time in a pentobarbital-induced sleep model. This hypnotic effect of AR-001 was significantly inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). As a result of immunohistochemistry, AR-001 was shown to increase neural activity in the sleep-promoting region, ventrolateral preoptic nucleus (VLPO), and decrease neural activity in the wake-promoting region, basal forebrain (BF), and lateral hypothalamus (LH), and that these effects of AR-001 were significantly inhibited by DPCPX treatment. In addition, AR-001 increased adenosine A1 receptor mRNA levels in the hypothalamus. In conclusion, this study suggests that AR-001 has a hypnotic effect, at least partially, through adenosine A1 receptor and may have therapeutic potential for insomnia.

Hit-to-Lead Optimization of Heterocyclic Carbonyloxycarboximidamides as Selective Antagonists at Human Adenosine A3 Receptor.

Antagonism of the human adenosine A3 receptor (hA3R) has potential therapeutic application. Alchemical relative binding free energy calculations of K18 and K32 suggested that the combination of a 3-(2,6-dichlorophenyl)-isoxazolyl group with 2-pyridinyl at the ends of a carbonyloxycarboximidamide group should improve hA3R affinity. Of the 25 new analogues synthesized, 37 and 74 showed improved hA3R affinity compared to K18 (and K32). This was further improved through the addition of a bromine group to the 2-pyridinyl at the 5-position, generating compound 39. Alchemical relative binding free energy calculations, mutagenesis studies and MD simulations supported the compounds' binding pattern while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, so highlighting the importance of rigidification of the carbonyloxycarboximidamide moiety. MD simulations highlighted the importance of rigidification of the carbonyloxycarboximidamide, while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, which was supported through mutagenesis studies 39 also selectively antagonized endogenously expressed hA3R in nonsmall cell lung carcinoma cells, while pharmacokinetic studies indicated low toxicity enabling in vivo evaluation. We therefore suggest that 39 has potential for further development as a high-affinity hA3R antagonist.

Adenosine triggers early astrocyte reactivity that provokes microglial responses and drives the pathogenesis of sepsis-associated encephalopathy in mice

Molecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic Gi protein-coupled A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial reaction, ameliorating blood-brain barrier disruption, peripheral immune cell infiltration, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as an important pathway driving SAE and highlight the potential of targeting A1ARs for therapeutic intervention.

Adenosine kinase protects against acetaminophen-induced acute liver injury by activating autophagy in hepatocytes

Acute liver injury (ALI) is a common life-threatening condition with a high mortality rate due to liver disease-related death. However, current therapeutic interventions for ALI remain ineffective, and the development of effective novel therapies is urgently needed. Liver samples from patients with drug-induced ALI were collected to detect adenosine kinase (ADK) expression. Male C57BL/6 J mice, hepatocyte-specific ADK knockout (ADKHKO) mice, and their controls (ADKf/f) were exposed to acetaminophen (APAP) and other treatments to investigate the mechanisms of APAP-related ALI. ADK expression was significantly decreased in APAP-injured livers. Hepatocyte-specific ADK deficiency exacerbated APAP-induced ALI, while a gain-of-function approach delivering AAV-ADK, markedly alleviated APAP-induced ALI, as indicated by changes in alanine aminotransferases (ALT) levels, aspartate aminotransferase (AST) levels, neutrophil infiltration and hepatocyte death. This study showed that ADK played a critical role in ALI by activating autophagy through two signaling pathways, the adenosine monophosphate-activated protein kinase (AMPK)-mTOR pathway and the adenosine receptor A1 (ADORA1)-Akt-mTOR pathway. Furthermore, we found that metformin upregulated ADK expression in hepatocytes and protected against APAP-induced ALI. These results demonstrate that ADK is critical in protecting against APAP-induced ALI and that developing therapeutics targeting ADK-adenosine-ADORA1 is a new approach for ALI treatment. Metformin is a potential candidate for preventing ALI by upregulating ADK.Graphical

Antidepressant effects of selective adenosine receptor antagonists targeting the A1 and A2A receptors administered jointly with NMDA receptor ligands: behavioral, biochemical and molecular investigations in mice

BackgroundThe objective of the study was to ascertain the antidepressant potential of the co-administration of NMDA receptor ligands and selective adenosine A1 and A2A receptor antagonists.MethodsThe forced swim test (FST) and spontaneous locomotor activity test were carried out in adult male naïve mice. Before the behavioral testing, animals received DPCPX (a selective adenosine A1 receptor antagonist, 1 mg/kg) or istradefylline (a selective adenosine A2A receptor antagonist, 0.5 mg/kg) in combination with L–701,324 (a potent NMDA receptor antagonist, 1 mg/kg), D–cycloserine (a partial agonist at the glycine recognition site of NMDA receptor, 2.5 mg/kg), CGP 37849 (a competitive NMDA receptor antagonist, 0.3 mg/kg) or MK–801 (a non-competitive NMDA receptor antagonist, 0.05 mg/kg). Additionally, serum BDNF level and the mRNA level of the Adora1, Comt, and Slc6a15 genes in the murine prefrontal cortex were determined.ResultsThe obtained results showed that DPCPX and istradefylline administered jointly with NMDA receptor ligands (except for DPCPX + D–cycloserine combination) produced an antidepressant effect in the FST in mice without enhancement in spontaneous motility of animals. An elevation in BDNF concentration was noted in the D–cycloserine-treated group. Adora1 expression increased with L–701,324, DPCPX + D–cycloserine, and DPCPX + CGP 37849, while D–cycloserine, CGP 37849, and MK–801 led to a decrease. Comt mRNA levels dropped with DPCPX + L–701,324, istradefylline + L–701,324/CGP 37849 but increased with D–cycloserine, MK–801, CGP 37849 and DPCPX + MK–801/ CGP 37849. Slc6a15 levels were reduced by D–cycloserine, DPCPX + L–701,324 but rose with DPCPX + CGP 37849/MK–801 and istradefylline + D–cycloserine/MK–801/CGP 37849.ConclusionOur study suggests that selective antagonists of adenosine receptors may enhance the antidepressant efficacy of NMDA receptor ligands highlighting a potential synergistic interaction between the adenosinergic and glutamatergic systems. Wherein, A2A receptor antagonists are seen as more promising candidates in this context. Given the intricate nature of changes in BDNF levels and the expression of Adora1, Comt, and Slc6a15 seen after drug combinations exerting antidepressant properties, further research and integrative approaches are crucial understand better the mechanisms underlying their antidepressant action.

Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

Delta-9-Tetrahydrocannabinol Blocks Bone Marrow-Derived Macrophage Differentiation through Elimination of Reactive Oxygen Species.

Macrophages are vital components of the immune system and serve as the first line of defense against pathogens. Macrophage colony-stimulating factor (M-CSF) induces macrophage differentiation from bone marrow-derived cells (BMDCs). Δ9-tetrahydrocannabiol (THC), a phytocannabinoid from the Cannabis plant, has profound anti-inflammatory properties with significant effects on myeloid cells. To investigate the effect of THC on macrophage differentiation, we cultured BMDCs with M-CSF in the presence of THC. Interestingly, THC markedly blocked the differentiation of BMDCs into CD45 + CD11b + F4/80+ macrophages. The effect of THC was independent of cannabinoid receptors CB1, and CB2, as well as other potential receptors such as GPR18, GPR55, and Adenosine 2A Receptor. RNA-seq analysis revealed that the THC-treated BMDCs displayed a significant increase in the expression of NRF2-ARE-related genes. KEGG pathway analysis revealed that the expression profiles of THC-treated cells correlated with ferroptosis and glutathione metabolism pathways. Fluorescence-based labile iron assays showed that the THC-treated BMDCs had significantly increased iron levels. Finally, THC-exposed BMDCs showed decreased levels of intracellular ROS. THC has the unique molecular property to block the Fenton Reaction, thus preventing the increase in intracellular ROS that is normally induced by high iron levels. Together, these studies demonstrated that THC blocks M-CSF-induced macrophage differentiation by inhibiting ROS production through both the induction of NRF2-ARE-related gene expression and the prevention of ROS formation via the Fenton Reaction.

Development of Preladenant-Based Radiotracers for Imaging A2AR in Tumors.

Activation of the adenosine 2A receptor (A2AR) can lead to tumor immunosuppression, which results in poor prognosis of immunotherapy. The aim of this study was to design novel 18F-labeled probes ([18F]F-PFP2 and [18F]F-PFP4) to visualize A2AR in the tumor. The uptake of radioprobes in A2AR-negative 4T1 breast tumor was lower than that of A2AR-positive B16F10 melanoma at 1 h p.i. (1.22 ± 0.36% ID/g vs 2.80 ± 0.72% ID/g), 2 h p.i. (1.09 ± 0.20% ID/g vs 2.93 ± 0.76% ID/g) and 3 h p.i. (0.89 ± 0.27% ID/g vs 2.73 ± 0.58% ID/g), respectively. B16F10 lung metastasis models were employed to expand the application scenarios, observing significantly higher uptake of [18F]F-PFP2 in metastatic lesions compared to normal lung tissue (5.55 ± 2.18% ID/g vs 1.89 ± 0.65% ID/g, tumor/lung ratio ∼3). It is given that [18F]F-PFP2 might lay the foundation for establishing an A2AR-targeted imaging evaluation system for tumors, which will provide more precise guidance for personalized treatment.

N6-cyclohexyladenosine is better than meperidine and buspirone at suppressing metabolism during TTM32 but does not improve outcome after cardiac arrest

N6-clyclohexyladenosine (CHA) is an adenosine A1 receptor agonist that inhibits thermogenesis. Cardiovascular side effects however, limit use of CHA as a therapeutic. We and others have shown that this can be reversed by administering 8-p-(sulfophenyl)theophylline (8-SPT), a nonspecific antagonist that does not cross the BBB. Other evidence shows that CNS actions of CHA may contribute to bradycardia through enhanced vagal tone and other mechanisms. Here we test the hypothesis that 8-SPT pretreatment alone is sufficient to prevent hypotension caused by CHA. To test this hypothesis, we pretreated rats with 8-SPT alone, and in combination with other antagonists to test the hypothesis that direct action of CHA on the heart is the primary mechanism by which CHA induces bradycardia and hypotension. Results show that pretreatment with 8-SPT alone is not sufficient to prevent CHA-induced hypotension. Pretreatment with 8-SPT or atropine alone did not prevent the fall in mean arterial pressure (MAP) and heart rate (HR), however, pretreatment with 8-SPT (25 mg/kg) and atropine (1 mg/kg) 15 min before CHA (1 mg/kg) preserves MAP and HR baseline values after CHA administration. We next asked if blood pressure was managed during the transition into a hypometabolic state, would prolong CHA-mediated inhibition of metabolism after cardiac arrest improve outcome better than anti-shivering medications meperidine and buspirone. We found that CHA-mediated hypotension can be mitigated by pretreatment with atropine and 8-SPT. This combination administered after cardiac arrest facilitated temperature management and metabolic suppression better than meperidine and buspirone, however, did not improve survival.

Analgesia by fascia manipulation is mediated by peripheral and spinal adenosine A1 receptor in a mouse model of peripheral inflammation

The role of adenosine receptors in fascial manipulation-induced analgesia has not yet been investigated. The purpose of this study was to evaluate the involvement of the adenosine A1 receptor (A1R) in the antihyperalgesic effect of plantar fascia manipulation (PFM), specifically in mice with peripheral inflammation. Mice injected with Complete Freund’s Adjuvant (CFA) underwent behavioral, i.e. mechanical hyperalgesia and edema. The mice underwent PFM for either 3, 9 or 15 min. Response frequency to mechanical stimuli was then assessed at 24 and 96 h after plantar CFA injection. The adenosinergic receptors were assessed by systemic (intraperitoneal, i.p.), central (intrathecal, i.t.), and peripheral (intraplantar, i.pl.) administration of caffeine. The participation of the A1R was investigated using the 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1R subtype antagonist. PFM inhibited mechanical hyperalgesia induced by CFA injection and did not reduce paw edema. Furthermore, the antihyperalgesic effect of PFM was prevented by pretreatment of the animals with caffeine given by i.p., i.pl., and i.t. routes. In addition, i.pl. and i.t. administrations of DPCPX blocked the antihyperalgesia caused by PFM. These observations indicate that adenosine receptors mediate the antihyperalgesic effect of PFM. Caffeine’s inhibition of PFM-induced antihyperalgesia suggests that a more precise understanding of how fascia-manipulation and caffeine interact is warranted.

Structural Mass Spectrometry Captures Residue-Resolved Comprehensive Conformational Rearrangements of a G Protein-Coupled Receptor.

G protein-coupled receptor (GPCR) structural studies with in-solution spectroscopic approaches have offered distinctive insights into GPCR activation and signaling that highly complement those yielded from structural snapshots by crystallography or cryo-EM. While most current spectroscopic approaches allow for probing structural changes at selected residues or loop regions, they are not suitable for capturing a holistic view of GPCR conformational rearrangements across multiple domains. Herein, we develop an approach based on limited proteolysis mass spectrometry (LiP-MS) to simultaneously monitor conformational alterations of a large number of residues spanning both flexible loops and structured transmembrane domains for a given GPCR. To benchmark LiP-MS for GPCR conformational profiling, we studied the adenosine 2A receptor (A2AR) in response to different ligand binding (agonist/antagonist/allosteric modulators) and G protein coupling. Systematic and residue-resolved profiling of A2AR conformational rearrangements by LiP-MS precisely captures structural mechanisms in multiple domains underlying ligand engagement, receptor activation, and allostery, and may also reflect local conformational flexibility. Furthermore, these residue-resolution structural fingerprints of the A2AR protein allow us to readily classify ligands of different pharmacology and distinguish the G protein-coupled state. Thus, our study provides a new structural MS approach that would be generalizable to characterizing conformational transition and plasticity for challenging integral membrane proteins.

The cumulative analgesic effect of repeated electroacupuncture is modulated by Adora3 in the SCDH of mice with neuropathic pain.

Existing remedial approaches for relieving neuropathic pain (NPP) are challenging and open the way for alternative therapeutic measures such as electroacupuncture (EA). The mechanism underlying the antinociceptive effects of repeated EA sessions, particularly concerning the regulation of the Adora3 receptor and its associated enzymes, has remained elusive. This study used a mouse model of spared nerve injury (SNI) to explore the cumulative analgesic effects of repeated EA at ST36 (Zusanli) and its impact on Adora3 regulation in the spinal cord dorsal horn (SCDH). Forty-eight male mice underwent SNI surgery for induction of neuropathic pain and were randomly assigned to the SNI, SNI + 2EA, SNI + 4EA, and SNI + 7EA groups. Spinal cord (L4-L6) was sampled for immunofluorescence, adenosine (ADO) detection and for molecular investigations following repeated EA treatment. Following spared nerve injury (SNI), there was a significant decrease in mechanical withdrawal thresholds (PWTs) and thermal nociceptive withdrawal latency (TWL) in the ipsilateral hind paw on the third day post-surgery, while the contralateral hind paw PWTs showed no significant changes. On subsequent EA treatments, the SNI + EA groups led to a significant increase in pain thresholds (p < 0.05). Repeated EA sessions in SNI mice upregulated Adenosine A3 (Adora3) and cluster of differentiation-73 (CD73) expression while downregulating adenosine deaminase (ADA) and enhancing neuronal instigation in the SCDH. Colocalization analysis of Neun-treated cells revealed increased Adora3 expression, particularly in the SNI + 7EA group. In conclusion, cumulative electroacupuncture treatment reduced neuropathic pain by regulating Adora3 and CD73 expression, inhibiting ADA and most likely increasing neuronal activation in the SCDH. This study offers a promising therapeutic option for managing neuropathic pain, paving the way for further research.

Oncolytic Peptide-Nanoplatform Drives Oncoimmune Response and Reverses Adenosine-Induced Immunosuppressive Tumor Microenvironment.

The application of oncolytic peptides has become a powerful approach to induce complete and long-lasting remission in multiple types of carcinomas, as affirmed by the appearance of tumor-associated antigens and adenosine triphosphate (ATP) in large quantities, which jumpstarts the cancer-immunity cycle. However, the ATP breakdown product adenosine is a significant contributor to forming the immunosuppressive tumor microenvironment, which substantially weakens peptide-driven oncolytic immunotherapy. In this study, a lipid-coated micelle (CA@TLM) loaded with a stapled oncolytic peptide (PalAno) and an adenosine 2A receptor (A2AR) inhibitor (CPI-444) is devised to enact tumor-targeted oncolytic immunotherapy and to overcome adenosine-mediated immune suppression simultaneously. The CA@TLM micelle accumulates in tumors with high efficiency, and the acidic tumor microenvironment prompts the rapid release of PalAno and CPI-444. Subsequently, PalAno induces swift membrane lysis of tumor cells and the release of antigenic materials. Meanwhile, CPI-444 blocks the activation of the immunosuppressive adenosine-A2AR signaling pathway. This combined approach exhibits pronounced synergy at stalling tumor growth and metastasis in animal models for triple-negative breast cancer and melanoma, providing a novel strategy for enhanced oncolytic immunotherapy.

Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs).

A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the Emax of A3AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A3ARs. The chain length, functionality, and attachment position were varied to modulate A3AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's Emax and potency ([35S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A3AR PAM activity through rational design based on an extrahelical, lipidic binding site.

Lack of Interactions Between Alteplase/Tenecteplase and the Adenosine A1R/A3R Agonist AST-004.

AST-004, a small molecule agonist of the adenosine A1 and A3 receptors, is a potential cerebroprotectant for patients with acute stroke and is currently in clinical trials. Drug-drug interactions are critically important to assess in the context of acute stroke care. Lytic therapy with tPA (tissue-type plasminogen activator)-induced plasmin formation (alteplase) is the only available pharmacotherapy for acute stroke. Consequently, it is imperative to evaluate potential interactions between AST-004 and tPAs such as alteplase and tenecteplase. The interactions between AST-004 and tPAs were evaluated in 3 ways in preparation for AST-004 phase II trials. First, the metabolic stability of AST-004 was determined in the presence of alteplase and plasmin. Second, the potential for AST-004 to influence the thrombolytic efficacy of alteplase and tenecteplase was evaluated with an in vitro assay system utilizing a fluorogenic substrate of plasmin. Finally, the potential for AST-004 to influence the thrombolytic efficacy of alteplase was also determined with an in vitro thrombolysis assay of human blood thrombi. Neither alteplase nor plasmin affected the stability of AST-004 in vitro. In 2 different in vitro systems, AST-004 had no effect on the ability of alteplase or tenecteplase to generate plasmin, and AST-004 had no effect on the thrombolytic efficacy of alteplase to lyse blood clots in human blood. These studies indicate that there will be no interactions between AST-004 and tPAs such as alteplase or tenecteplase in patients with stroke undergoing thrombolytic therapy.

Targeting Adenosine A2b Receptor Promotes Penile Rehabilitation of Refractory Erectile Dysfunction.

The mechanisms of adenosine and specific adenosine receptor subtypes in promoting penile rehabilitation remain unclear. Single-cell RNA sequencing of human corpus cavernosum, adenosine deaminase (ADA) and adenosine receptors knock-out mice (ADA-/-, A1-/-, A2a-/-, A2b-/-, and A3-/-), and primary corpus cavernosum smooth muscle cellsare used to determine receptor subtypes responsible for adenosine-induced erection. Three rat models are established to characterize refractory erectile dysfunction (ED): age-related ED, bilateral cavernous nerve crush related ED (BCNC), and diabetes mellitus-induced ED. In single-cell RNA sequencing data, the corpus cavernosum of ED patients show a decrease in adenosine A1, A2a and A2b receptors. In vivo, A2b receptor knock-out abolishes adenosine-induced erection but not that of A1, A2a, or A3 receptor. Under hypoxic conditions in vitro, activating the A2b receptor increases HIF-1α and decreases PDE5 expression. In refractory ED models, activating the A2b receptor with Bay 60-6583 improves erectile function and down-regulates HIF-1α and TGF-β. Administering Dipyridamole (40mgKg-1) to BCNC rats improve penile adenosine levels and erectile function. Our study reveals that the A2b receptor mediates adenosine-induced penile erection. Activating the A2b receptor promotes penile rehabilitation of refractory ED by alleviating hypoxia and fibrosis.

2-Substituted (N)-Methanocarba A3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9-11, 13, 14, 19, 22, 23, 27, 29, 31, and 34, lacking a spacer, had human (h) A3AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A3AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (7, 8 > 3c, 3d > 3b). However, 2-aryl-4'-truncated derivatives had greatly reduced hA3AR affinity, even containing affinity-enhancing N 6-dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A3AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA3AR miniGαi recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy (E max) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A3AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a-3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A3AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A3AR agonist SAR for (N)-methanocarba adenosines.

Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Chronic Caffeine Consumption, Alone or Combined with Agomelatine or Quetiapine, Reduces the Maximum EEG Peak, As Linked to Cortical Neurodegeneration, Ovarian Estrogen Receptor Alpha, and Melatonin Receptor 2

RationaleEvidence of the effects of chronic caffeine (CAFF)-containing beverages, alone or in combination with agomelatine (AGO) or quetiapine (QUET), on electroencephalography (EEG), which is relevant to cognition, epileptogenesis, and ovarian function, remains lacking. Estrogenic, adenosinergic, and melatonergic signaling is possibly linked to the dynamics of these substances.ObjectivesThe brain and ovarian effects of CAFF were compared with those of AGO + CAFF and QUET + CAFF. The implications of estrogenic, adenosinergic, and melatonergic signaling and the brain-ovarian crosstalk were investigated.MethodsAdult female rats were administered AGO (10 mg/kg), QUET (10 mg/kg), CAFF, AGO + CAFF, or QUET + CAFF, once daily for 8 weeks. EEG, estrous cycle progression, and microstructure of the brain and ovaries were examined. Brain and ovarian 17β-estradiol (E2), antimullerian hormone (AMH), estrogen receptor alpha (E2Rα), adenosine receptor 2A (A2AR), and melatonin receptor 2 (MT2R) were assessed.ResultsCAFF, alone or combined with AGO or QUET, reduced the maximum EEG peak, which was positively linked to ovarian E2Rα, negatively correlated to cortical neurodegeneration and ovarian MT2R, and associated with cystic ovaries. A large corpus luteum emerged with AGO + CAFF and QUET + CAFF, antagonizing the CAFF-mediated increased ovarian A2AR and reduced cortical E2Rα. AGO + CAFF provoked TTP delay and increased ovarian AMH, while QUET + CAFF slowed source EEG frequency to δ range and increased brain E2.ConclusionsCAFF treatment triggered brain and ovarian derangements partially antagonized with concurrent AGO or QUET administration but with no overt affection of estrus cycle progression. Estrogenic, adenosinergic, and melatonergic signaling and brain-ovarian crosstalk may explain these effects.

Activating A1 adenosine receptor signaling boosts early pulmonary neutrophil recruitment in aged mice in response to Streptococcus pneumoniae infection

BackgroundStreptococcus pneumoniae (pneumococcus) is a leading cause of pneumonia in older adults. Successful control of pneumococci requires robust pulmonary neutrophil influx early in infection. However, aging is associated with aberrant neutrophil recruitment and the mechanisms behind that are not understood. Here we explored how neutrophil recruitment following pneumococcal infection changes with age and the host pathways regulating this.ResultsFollowing pneumococcal infection there was a significant delay in early neutrophil recruitment to the lungs of aged mice. Neutrophils from aged mice showed defects in trans-endothelial migration in vitro compared to young controls. To understand the pathways involved, we examined immune modulatory extracellular adenosine (EAD) signaling, that is activated upon cellular damage. Signaling through the lower affinity A2A and A2B adenosine receptors had no effect on neutrophil recruitment to infected lungs. In contrast, inhibition of the high affinity A1 receptor in young mice blunted neutrophil recruitment to the lungs following infection. A1 receptor inhibition decreased expression of CXCR2 on circulating neutrophils, which is required for trans-endothelial migration. Indeed, A1 receptor signaling on neutrophils was required for their ability to migrate across endothelial cells in response to infection. Aging was not associated with defects in EAD production or receptor expression on neutrophils. However, agonism of A1 receptor in aged mice rescued the early defect in neutrophil migration to the lungs and improved control of bacterial burden.ConclusionsThis study suggests age-driven defects in EAD damage signaling can be targeted to rescue the delay in pulmonary neutrophil migration in response to bacterial pneumonia.