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The liver enzyme cytochrome P450 1A2 (CYP1A2) is responsible for 90% of caffeine metabolism, while caffeine exerts many of its effects via antagonist binding to adenosine A2a receptors (ADORA2A). This study aimed to examine whether functional single nucleotide polymorphisms (SNPs) in 1976T > C (ADORA2A; rs5751876) and −163C > A (CYP1A2; rs762551) influence the effect of caffeine on the postprandial glucose (GLU) response to a carbohydrate meal. We report that individuals with the 1976T > C CC, but not CT/TT genotypes display elevated GLU levels after consuming caffeine and carbohydrate (CHO + CAFF) versus carbohydrate only (CHO). The GLU area under the curve (AUC) was also greater during the CHO + CAFF condition compared to the CHO condition in CC, but not the CT/TT genotypes. The −163C > A AC/CC, but not AA, genotypes displayed greater GLU concentrations 60-min post meal during CHO + CAFF versus CHO. Our data suggest that caffeine-induced impairments in postprandial glycaemia are related to 1976T > C and −163C > A SNPs.

Evidence has shown that both sleep loss and daily caffeine intake can induce changes in grey matter (GM). Caffeine is frequently used to combat sleepiness and impaired performance caused by insufficient sleep. It is unclear (1) whether daily use of caffeine could prevent or exacerbate the GM alterations induced by 5-day sleep restriction (i.e. chronic sleep restriction, CSR), and (2) whether the potential impact on GM plasticity depends on individual differences in the availability of adenosine receptors, which are involved in mediating effects of caffeine on sleep and waking function. Thirty-six healthy adults participated in this double-blind, randomized, controlled study (age = 28.9 ± 5.2 y/; F:M = 15:21; habitual level of caffeine intake < 450 mg; 29 homozygous C/C allele carriers of rs5751876 of ADORA2A, an A 2A adenosine receptor gene variant). Each participant underwent a 9-day laboratory visit consisting of one adaptation day, 2 baseline days (BL), 5-day sleep restriction (5 h time-in-bed), and a recovery day (REC) after an 8-h sleep opportunity. Nineteen participants received 300 mg caffeine in coffee through the 5 days of CSR (CAFF group), while 17 matched participants received decaffeinated coffee (DECAF group). We examined GM changes on the 2nd BL Day, 5th CSR Day, and REC Day using magnetic resonance imaging and voxel-based morphometry. Moreover, we used positron emission tomography with [ 18 F]-CPFPX to quantify the baseline availability of A 1 adenosine receptors (A 1 R) and its relation to the GM plasticity. The results from the voxel-wise multimodal whole-brain analysis on the Jacobian-modulated T1-weighted images controlled for variances of cerebral blood flow indicated a significant interaction effect between caffeine and CSR in four brain regions: (a) right temporal-occipital region, (b) right dorsomedial prefrontal cortex (DmPFC), (c) left dorsolateral prefrontal cortex (DLPFC), and (d) right thalamus. The post-hoc analyses on the signal intensity of these GM clusters indicated that, compared to BL, GM on the CSR day was increased in the DECAF group in all clusters  but decreased in the thalamus, DmPFC, and DLPFC in the CAFF group. Furthermore, lower baseline subcortical A 1 R availability predicted a larger GM reduction in the CAFF group after CSR of all brain regions except for the thalamus. In conclusion, our data suggest an adaptive GM upregulation after 5-day CSR, while concomitant use of caffeine instead leads to a GM reduction. The lack of consistent association with individual A 1 R availability may suggest that CSR and caffeine affect thalamic GM plasticity predominantly by a different mechanism. Future studies on the role of adenosine A 2A receptors in CSR-induced GM plasticity are warranted.

Sleep control is ascribed to a two-process model, a widely accepted concept that posits homoeostatic drive and a circadian process as the major sleep-regulating factors. Cognitive and emotional factors also influence sleep–wake behaviour; however, the precise circuit mechanisms underlying their effects on sleep control are unknown. Previous studies suggest that adenosine has a role affecting behavioural arousal in the nucleus accumbens (NAc), a brain area critical for reinforcement and reward. Here, we show that chemogenetic or optogenetic activation of excitatory adenosine A2A receptor-expressing indirect pathway neurons in the core region of the NAc strongly induces slow-wave sleep. Chemogenetic inhibition of the NAc indirect pathway neurons prevents the sleep induction, but does not affect the homoeostatic sleep rebound. In addition, motivational stimuli inhibit the activity of ventral pallidum-projecting NAc indirect pathway neurons and suppress sleep. Our findings reveal a prominent contribution of this indirect pathway to sleep control associated with motivation.

Determination of the crystal structure of the human A 2A adenosine receptor in complex with an inverse-agonist antibody shows that the allosteric site of the receptor inhibits agonist binding and that the antibody locks the receptor in an inactive conformation. GPCR modulation by an antibody G-protein-coupled receptors are the largest class of cell-surface receptors, and many of them are considered drug targets. The crystal structure of the human A2A adenosine receptor in complex with a mouse antibody fragment has now been determined. This structure includes the intracellular loop 3, critical for G-protein binding. In addition, the use of an antibody fragment to stabilize the receptor in an inactive conformation suggests a new strategy to modulate the activity of G-protein-coupled receptors. G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A 2A adenosine receptor (A 2A AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain 14 . Here we report the raising of a mouse monoclonal antibody against human A 2A AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A 2A AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A 2A AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure 1 and to CDR-3 of the nanobody in the active β 2 -adrenergic receptor structure 2 , but locks A 2A AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A ₂A/A ₂B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A ₂A⁻/⁻ mice were strongly protected against tumor metastasis, indicating that host A ₂A receptors enhanced tumor metastasis. A ₂A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A ₂A blockade was due to enhanced NK cell function. Interestingly, A ₂B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73 ⁺ tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A ₂A or A ₂B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A ₂A/A ₂B receptor antagonists have already entered clinical trials in other therapeutic settings.

Extracellular vesicle-driven cancer metastasis represents a therapeutic challenge due to the lack of effective blocking drugs. Our study shows that activation of the P2X7 receptor on colon carcinoma cells causes the release of vesicles carrying CD39 and CD73 ectonucleotidases. These vesicles increase ATP and adenosine levels and, when in vivo administered, significantly enhance colon carcinoma metastasis and circulating levels of vesicles after fourteen days from their injection. Blocking P2X7 prevents vesicular release and substantially reduces vesicle-mediated tumor spreading, positioning P2X7 as a promising therapeutic target for inhibiting extracellular vesicle-mediated dissemination in colon cancer. Additionally, these vesicles upregulate the expression of P2X7 and A2A receptors within the metastatic niche. Antagonists of P2X7 and A2A used alone or in combination effectively inhibit tumor growth in vivo, decreasing metastasis engraftment and IL-17 and IL-23 release. Interestingly, the levels of both cytokines were also reduced by combined P2X7 and A2A blockade in non-tumor-bearing mice. Moreover, P2X7 and A2A upraise in metastatic and APC -mutated colon carcinoma patients and in Apc -disrupted rats. Our findings shed light on the crosstalk of P2X7/CD73/CD39 and A2A in colon cancer metastasis. We propose a novel mechanism facilitating metastatic dissemination and an innovative therapeutic strategy to target receptor signaling and vesicular release.

Background Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC. Methods The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model. Results Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4 + Foxp3 + regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4 + Foxp3 + Tregs and enhanced the anti-tumor response of CD8 + T cells. Conclusions These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

Caffeine produces mild psychostimulant and sometimes anxiogenic effects by antagonizing adenosine at A 1 and A 2A receptors, and perhaps through interactions with other transmitter systems. Adenosine receptors are colocalized and functionally interact with dopamine receptors in the brain. Thus, functional polymorphisms in the genes for either adenosine or dopamine receptors may affect responses to caffeine. In this study, we examined associations between self-reported anxiogenic effects of caffeine and variation in the genes for A 2A ( ADORA2A ) and DRD 2 ( DRD2 ) receptors. Healthy male and female individuals ( n =102), who consumed less than 300 mg caffeine per week, ingested capsules containing 0, 50, 150, and 450 mg caffeine under double-blind conditions in four separate experimental sessions. Subjective anxiety was measured before and at repeated times after capsules were consumed. At the 150 mg dose of caffeine, we found a significant association between caffeine-induced anxiety (Visual Analog Scales, VAS) and ADORA2A rs5751876 (1976C/T), rs2298383 (intron 1a) and rs4822492 (3′-flank), and DRD2 rs1110976 (intron 6). Caffeine-induced anxiety (VAS) was also associated with two-loci interactions of selected ADORA2A and DRD2 polymorphisms. The lowest dose of caffeine did not increase ratings of anxiety while the highest dose increased anxiety in the majority of subjects. These findings provide support for an association between an ADORA2A polymorphism and self-reported anxiety after a moderate dose of caffeine. It is likely that other ADORA2A and DRD2 polymorphisms also contribute to responses to caffeine.