Clinical and Biologic Correlates of ADORA2A Transcriptomic Expression in Cancer.

ADORA2A (adenosine A2a receptor) and ADORA2B propagate immunoregulatory signals, including restricting both innate and adaptive immunity, though recent data also suggest a tumor suppressor effect in certain settings. We evaluated the RNA expression from 514 tumors in a clinical-grade laboratory; 489 patients with advanced/metastatic disease had clinical outcome correlates. Transcript expression was standardized to internal housekeeping genes and ranked (0-100 scale) relative to 735 specimens from 35 different cancer types. Transcript abundance rank values were defined as "low/moderate" (0-74) or "high" (75-100) percentile RNA expression ranks. Overall, 20.8% of tumors had high ADORA2A (≥75 percentile RNA rank). The greatest proportion of high ADORA2A expressors was found in neuroendocrine and breast cancers and sarcomas, whereas the lowest was found in colorectal and ovarian cancers, albeit with patient-to-patient variability. In multivariable logistic regression analysis, there was a significant positive correlation between high ADORA2A RNA expression and a high expression of the immune checkpoint-related molecules PD-1 (p = 0.015), VISTA (p ≤ 0.001), CD38 (p = 0.031), and CD39 (p ≤ 0.001). In 217 immunotherapy-treated patients, high ADORA2A did not correlate significantly with progression-free (p = 0.51) or overall survival (OS) (p = 0.09) from the initiation of the checkpoint blockade. However, high versus not-high ADORA2A transcript expression correlated with longer OS from the time of advanced/metastatic disease (N = 489 patients; (HR 0.69 (95% CI 0.51-0.95) (p = 0.02)). Therefore, high ADORA2A transcript levels may be a favorable prognostic factor, unrelated to immunotherapy. Importantly, ascertaining co-expression patterns of ADORA2A with PD-1 and VISTA in individual tumors as a basis for the precision co-targeting of ADORA2A and these other checkpoint-related molecules warrants investigation in clinical trials.

Adenosine A2A Receptor Blockade Provides More Effective Benefits at the Onset Rather than after Overt Neurodegeneration in a Rat Model of Parkinson's Disease.

Adenosine A2A receptor (A2AR) antagonists are the leading nondopaminergic therapy to manage Parkinson's disease (PD) since they afford both motor benefits and neuroprotection. PD begins with a synaptic dysfunction and damage in the striatum evolving to an overt neuronal damage of dopaminergic neurons in the substantia nigra. We tested if A2AR antagonists are equally effective in controlling these two degenerative processes. We used a slow intracerebroventricular infusion of the toxin MPP+ in male rats for 15 days, which caused an initial loss of synaptic markers in the striatum within 10 days, followed by a neuronal loss in the substantia nigra within 30 days. Interestingly, the initial loss of striatal nerve terminals involved a loss of both dopaminergic and glutamatergic synaptic markers, while GABAergic markers were preserved. The daily administration of the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) in the first 10 days after MPP+ infusion markedly attenuated both the initial loss of striatal synaptic markers and the subsequent loss of nigra dopaminergic neurons. Strikingly, the administration of SCH58261 (0.1 mg/kg, i.p. for 10 days) starting 20 days after MPP+ infusion was less efficacious to attenuate the loss of nigra dopaminergic neurons. This prominent A2AR-mediated control of synaptotoxicity was directly confirmed by showing that the MPTP-induced dysfunction (MTT assay) and damage (lactate dehydrogenase release assay) of striatal synaptosomes were prevented by 50 nM SCH58261. This suggests that A2AR antagonists may be more effective to counteract the onset rather than the evolution of PD pathology.

A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis.

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

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.

Adora2A downregulation promotes caffeine neuroprotective effect against LPS-induced neuroinflammation in the hippocampus.

Caffeine has been extensively studied in the context of CNS pathologies as many researchers have shown that consuming it reduces pro-inflammatory biomarkers, potentially delaying the progression of neurodegenerative pathologies. Several lines of evidence suggest that adenosine receptors, especially A1 and A2A receptors, are the main targets of its neuroprotective action. We found that caffeine pretreatment 15 min before LPS administration reduced the expression of Il1b in the hippocampus and striatum. The harmful modulation of caffeine-induced inflammatory response involved the downregulation of the expression of A2A receptors, especially in the hippocampus. Caffeine treatment alone promoted the downregulation of the adenosinergic receptor Adora2A; however, this promotion effect was reversed by LPS. Although administering caffeine increased the expression of the enzymes DNA methyltransferases 1 and 3A and decreased the expression of the demethylase enzyme Tet1, this effect was reversed by LPS in the hippocampus of mice that were administered Caffeine + LPS, relative to the basal condition; no significant differences were observed in the methylation status of the promoter regions of adenosine receptors. Finally, the bioinformatics analysis of the expanded network demonstrated the following results: the Adora2B gene connects the extended networks of the adenosine receptors Adora1 and Adora2A; the Mapk3 and Esr1 genes connect the extended Adora1 network; the Mapk4 and Arrb2 genes connect the extended Adora2A network with the extended network of the proinflammatory cytokine Il1ß. These results indicated that the anti-inflammatory effects of acute caffeine administration in the hippocampus may be mediated by a complex network of interdependencies between the Adora2B and Adora2A genes.

Blockade of endothelial adenosine receptor 2 A suppresses atherosclerosis in vivo through inhibiting CREB-ALK5-mediated endothelial to mesenchymal transition.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2 A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe-/- mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe-/- mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFß-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.

Low Density Lipoprotein Cholesterol Decreases the Expression of Adenosine A2A Receptor and Lipid Rafts-Protein Flotillin-1: Insights on Cardiovascular Risk of Hypercholesterolemia.

High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A2A receptor (A2AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. A2AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. A2AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in A2AR and Flotillin-1 expression. We suggest that LDL-C affects A2AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.

18F-Labeled PET Tracers Specific for Adenosine A2A Receptor: Design, Synthesis, and Biological Evaluation.

By modifying the structures of targeted A2AR antagonists and tracers, novel compounds 3, 7a, 9, 12c, and BIBD-399 were designed and synthesized. In vitro inhibition experiments demonstrated that 3, 12c, and BIBD-399 have high affinity for A2AR. [18F]3 and [18F]BIBD-399 were successfully synthesized. In terms of biological distribution, the brain uptake of [18F]MNI-444 exhibits greater than that of [18F]3 and [18F]BIBD-399. PET imaging shows that [18F]3 is off-target in the brain, while [18F]BIBD-399 and [18F]MNI-444 can be specifically imaged in regions with high A2AR expression. Differently, [18F]BIBD-399 could quickly reach equilibrium in the targeted region within 10 min after administration, while [18F]MNI-444 shows a slowly increasing trend within 2 h of administration. [18F]BIBD-399 is mainly metabolized by the liver and kidney, and there is no obvious defluorination in vivo. Additional in vitro autoradiography showed that the striatal signals of [18F]BIBD-399 and [18F]MNI-444 were inhibited by the A2AR antagonist SCH442416 but not by the A1R antagonist DPCPX, demonstrating the high A2AR binding specificity of [18F]BIBD-399. Molecular docking further confirms the high affinity of MNI-444 and BIBD-399 for A2AR. Further tMCAo imaging showed that [18F]BIBD-399 can sensitively distinguish between infarcted and noninfarcted sides, a capability not observed with [18F]MNI-444. Given its pharmacokinetic properties and the ability to identify lesion regions, [18F]BIBD-399 has potential advantages in monitoring A2AR changes, meriting further clinical investigation.

Subtle Structural Changes across the Boundary between A2AR/A2BR Dual Antagonism and A2BR Antagonism: A Novel Class of 2-Aminopyrimidine-Based Derivatives.

Aberrantly elevated adenosine in the tumor microenvironment exerts its immunosuppressive functions through adenosine receptors A2AR and A2BR. Antagonism of A2AR and A2BR has the potential to suppress tumor growth. Herein, we report a systemic assessment of the effects of an indole modification at position 4, 5, 6, or 7 on both A2AR/A2BR activity and selectivity of novel 2-aminopyrimidine compounds. Substituting indole at the 4-/5-position produced potent A2AR/A2BR dual antagonism, whereas the 6-position of indole substitution gave highly selective A2BR antagonism. Molecular dynamics simulation showed that the 5-cyano compound 7ai had a lower binding free energy than the 6-cyano compound 7aj due to water-bridged hydrogen bond interactions with E169 or F168 in A2AR. Of note, dual A2AR/A2BR antagonism by compound 7ai can profoundly promote the activation and cytotoxic function of T cells. This work provided a strategy for obtaining novel dual A2AR/A2BR or A2BR antagonists by fine-tuning structural modification.

Radiosynthesis and In Vitro Evaluation of [11C]tozadenant as Adenosine A2A Receptor Radioligand.

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A2A receptor (A2AR) antagonist and a promising lead structure for the development of A2AR-selective positron emission tomography (PET) probes. Although several 18F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with 18F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-11C]tozadenant ([11C]tozadenant) by O-methylation with [11C]methyl iodide followed by acidic deprotection. This approach afforded [11C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50-60 GBq/µmol (1300-1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant.

A novel A2a adenosine receptor inhibitor effectively mitigates hepatic fibrosis in a metabolic dysfunction-associated steatohepatitis mouse model.

Hepatic fibrosis exacerbates mortality and complications in progressive metabolic dysfunction-associated steatohepatitis (MASH). The role of the adenosine 2A receptor (A2aAR) in hepatic fibrosis within the context of MASH remains uncertain. This study aims to elucidate the involvement of the A2aAR signaling pathway and the efficacy of a novel potent A2aAR antagonist in treating hepatic fibrosis in MASH-induced mice fed a chlorine-deficient, L-amino acid-defined, high fat diet (CDAHFD). A2aAR overexpression in LX-2 cells increased fibrosis markers, whereas the known A2aAR antagonist, ZM241385, decreased these markers. A novel A2aAR antagonist, RAD11, not only attenuated fibrosis progression but also exhibited greater inhibition of the A2aAR signaling pathway compared to ZM241385 in mice with MASH, activated primary hepatocytes, and LX-2 cells. RAD11 exhibited a dual antifibrotic mechanism by targeting both activated HSCs and hepatocytes. Its superior antifibrotic efficacy over ZM241385 in the MASH condition stems from its ability to suppress A2aAR-mediated signaling, inhibit HSC activation, reduce hepatic lipogenesis in hepatocytes, and mitigate lipid accumulation-induced oxidative stress-mediated liver damage. This study has shed light on the relationship between A2aAR signaling and hepatic fibrosis, presenting RAD11 as a potent therapeutic agent for managing MASH and hepatic fibrosis.

CD8+ T cells sustain antitumor response by mediating crosstalk between adenosine A2A receptor and glutathione/GPX4.

Antitumor responses of CD8+ T cells are tightly regulated by distinct metabolic fitness. High levels of glutathione (GSH) are observed in the majority of tumors, contributing to cancer progression and treatment resistance in part by preventing glutathione peroxidase 4-dependent (GPX4-dependent) ferroptosis. Here, we show the necessity of adenosine A2A receptor (A2AR) signaling and the GSH/GPX4 axis in orchestrating metabolic fitness and survival of functionally competent CD8+ T cells. Activated CD8+ T cells treated ex vivo with simultaneous inhibition of A2AR and lipid peroxidation acquire a superior capacity to proliferate and persist in vivo, demonstrating a translatable means to prevent ferroptosis in adoptive cell therapy. Additionally, we identify a particular cluster of intratumoral CD8+ T cells expressing a putative gene signature of GSH metabolism (GMGS) in association with clinical response and survival across several human cancers. Our study addresses a key role of GSH/GPX4 and adenosinergic pathways in fine-tuning the metabolic fitness of antitumor CD8+ T cells.

Canagliflozin protects against hyperglycemia-induced cerebrovascular injury by preventing blood-brain barrier (BBB) disruption via AMPK/Sp1/adenosine A2A receptor.

Diabetes mellitus causes brain microvascular endothelial cell (MEC) damage, inducing dysfunctional angiogenic response and disruption of the blood-brain barrier (BBB). Canagliflozin is a revolutionary hypoglycemic drug that exerts neurologic and/or vascular-protective effects beyond glycemic control; however, its underlying mechanism remains unclear. In the present study, we hypothesize that canagliflozin ameliorates BBB permeability by preventing diabetes-induced brain MEC damage. Mice with high-fat diet/streptozotocin-induced diabetes received canagliflozin for 8 weeks. We assessed vascular integrity by measuring cerebrovascular neovascularization indices. The expression of specificity protein 1 (Sp1), as well as tight junction proteins (TJs), phosphorylated AMP-activated protein kinase (p-AMPK), and adenosine A2A receptors was examined. Mouse brain MECs were grown in high glucose (30 mM) to mimic diabetic conditions. They were treated with/without canagliflozin and assessed for migration and angiogenic ability. We also performed validation studies using AMPK activator (AICAR), inhibitor (Compound C), Sp1 small interfering RNA (siRNA), and adenosine A2A receptor siRNA. We observed that cerebral pathological neovascularization indices were significantly normalized in mice treated with canagliflozin. Increased Sp1 and adenosine A2A receptor expression and decreased p-AMPK and TJ expression were observed under diabetic conditions. Canagliflozin or AICAR treatment alleviated these changes. However, this alleviation effect of canagliflozin was diminished again after Compound C treatment. Either Sp1 siRNA or adenosine A2A receptor siRNA could increase the expression of TJs. Luciferase reporter assay confirmed that Sp1 could bind to the adenosine A2A receptor gene promoter. Our study identifies the AMPK/Sp1/adenosine A2A receptor pathway as a treatment target for diabetes-induced cerebrovascular injury.

Effects of the adenosine A2A receptor antagonist KW6002 on the dopaminergic system, motor performance, and neuroinflammation in a rat model of Parkinson's disease.

Adenosine A2A-receptors (A2AR) and dopamine D2-receptors (D2R) are known to work together in a synergistic manner. Inhibiting A2ARs by genetic or pharmacological means can relief symptoms and have neuroprotective effects in certain conditions. We applied PET imaging to evaluate the impact of the A2AR antagonist KW6002 on D2R availability and neuroinflammation in an animal model of Parkinson's disease. Male Wistar rats with 6-hydroxydopamine-induced damage to the right striatum were given 3 mg/kg of KW6002 daily for 20 days. Motor function was assessed using the rotarod and cylinder tests, and neuroinflammation and dopamine receptor availability were measured using PET scans with the tracers [11C]PBR28 and [11C]raclopride, respectively. On day 7 and 22 following 6-OHDA injection, rats were sacrificed for postmortem analysis. PET scans revealed a peak in neuroinflammation on day 7. Chronic treatment with KW6002 significantly reduced [11C]PBR28 uptake in the ipsilateral striatum [normalized to contralateral striatum] and [11C]raclopride binding in both striata when compared to the vehicle group. These imaging findings were accompanied by an improvement in motor function. Postmortem analysis showed an 84% decrease in the number of Iba-1+ cells in the ipsilateral striatum [normalized to contralateral striatum] of KW6002-treated rats compared to vehicle rats on day 22 (p = 0.007), corroborating the PET findings. Analysis of tyrosine hydroxylase levels showed less dopaminergic neuron loss in the ipsilateral striatum of KW6002-treated rats compared to controls on day 7. These findings suggest that KW6002 reduces inflammation and dopaminergic neuron loss, leading to less motor symptoms in this animal model of Parkinson's disease.

Effects of miR-214 on adenosine A2A receptor and carboxymethyl chitosan nanoparticles on the function of keloid fibroblasts and their mechanisms.

This work prepared and investigated the impact of carboxymethyl chitosan nanoparticles (MC-NPs) on the proliferative capability of keloid fibroblasts (KFBs) while analyzing the mechanistic roles of miR-214 and adenosine A2A receptor (A2AR) in fibroblasts within hypertrophic scars. MC-NPs were synthesized through ion cross-linking, were characterized using transmission electron microscopy (TEM) and laser particle size scattering. The influence of MC-NPs on the proliferation capacity of KFBs was assessed using the MTT method. Changes in the expression levels of miR-214 and A2AR in KFBs, normal skin fibroblasts (NFBs), hypertrophic scar tissue, and normal skin tissue were analyzed. KFBs were categorized into anti-miR-214, anti-miR-NC, miR-214 mimics, miR-NC, si-A2AR, si-con, anti-miR-214+ si-con, and anti-miR-214+ si-A2AR groups. Bioinformatics target prediction was conducted to explore the interaction between miR-214 and A2AR. Real-time quantitative PCR and immunoblotting (WB) were employed to detect the expression levels of miR-214, A2AR, apoptotic protein Bax, and TGF-ß in different cells. Cell counting kit-8 (CCK8) and flow cytometry were employed to assess cell proliferation activity and apoptosis. The results indicated that MC-NPs exhibited spherical particles with an average diameter of 236.47 ± 4.98 nm. The cell OD value in the MC-NPs group was lower than that in KFBs (P < 0.05). The mRNA levels of miR-214 in KFBs and hypertrophic scar tissue were lower than those in NFBs and normal tissue (P < 0.001), while the mRNA and protein levels of A2AR were significantly elevated (P < 0.05). Compared to the control group and anti-miR-NC, the anti-miR-214 group showed significantly increased cell OD values and Bcl-2 protein expression (P < 0.001), decreased levels of apoptotic gene Bax protein, TGF-ß gene mRNA, and protein expression (P < 0.001). Continuous complementary binding sites were identified between miR-214 and A2AR. Compared to the control group, the si-A2AR group exhibited a significant decrease in A2AR gene mRNA and protein expression levels (P < 0.001), reduced cell viability (P < 0.001), increased apoptosis rate (P < 0.001), and a significant elevation in TGF-ß protein expression (P < 0.001). miR-214 targetedly regulated the expression of A2AR, inducing changes in TGF-ß content, promoting the proliferation of keloid fibroblasts, and inhibiting cell apoptosis.

Structural insight into the dual-antagonistic mechanism of AB928 on adenosine A2 receptors.

The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2-118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2-118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy.

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.

Optical Control of Adenosine A2A Receptor Using Istradefylline Photosensitivity.

In recent years, there has been growing interest in the potential therapeutic use of inhibitors of adenosine A2A receptors (A2AR) for the treatment of neurodegenerative diseases and cancer. Nevertheless, the widespread expression of A2AR throughout the body emphasizes the importance of temporally and spatially selective ligands. Photopharmacology is an emerging strategy that utilizes photosensitive ligands to attain high spatiotemporal precision and regulate the function of biomolecules using light. In this study, we combined photochemistry and cellular and in vivo photopharmacology to investigate the light sensitivity of the FDA-approved antagonist istradefylline and its potential use as an A2AR photopharmacological tool. Our findings reveal that istradefylline exhibits rapid trans-to-cis isomerization under near-UV light, and prolonged exposure results in the formation of photocycloaddition products. We demonstrate that exposure to UV light triggers a time-dependent decrease in the antagonistic activity of istradefylline in A2AR-expressing cells and enables real-time optical control of A2AR signaling in living cells and zebrafish. Together, these data demonstrate that istradefylline is a photoinactivatable A2AR antagonist and that this property can be utilized to perform photopharmacological experiments in living cells and animals.

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.

Crystal structure reveals the binding mode and selectivity of a photoswitchable ligand for the adenosine A2A receptor.

Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.