Exploring Biginelli-based scaffolds as A2B adenosine receptor antagonists: Unveiling novel structure-activity relationship trends, lead compounds, and potent colorectal anticancer agents.

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.

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.

Co-inhibition of adenosine 2b receptor and programmed death-ligand 1 promotes the recruitment and cytotoxicity of natural killer cells in oral squamous cell carcinoma.

Adenosine promotes anti-tumor immune responses by modulating the functions of T-cells and natural killer (NK) cells in the tumor microenvironment; however, the role of adenosine receptors in the progression of oral squamous cell carcinoma (OSCC) and its effects on immune checkpoint therapy remain unclear. In this study, we obtained the tumor tissues from 80 OSCC patients admitted at the Shandong University Qilu Hospital between February 2014 and December 2016. Thereafter, we detected the expression of adenosine 2b receptor (A2BR) and programmed death-ligand 1 (PD-L1) using immunohistochemical staining and analyzed the association between their expression in different regions of the tumor tissues, such as tumor nest, border, and paracancer stroma. To determine the role of A2BR in PD-L1 expression, CAL-27 (an OSCC cell line) was treated with BAY60-6583 (an A2BR agonist), and PD-L1 expression was determined using western blot and flow cytometry. Furthermore, CAL-27 was treated with a nuclear transcription factor-kappa B (NF-κ B) inhibitor, PDTC, to determine whether A2BR regulates PD-L1 expression via the NF-κ B signaling pathway. Additionally, a transwell assay was performed to verify the effect of A2BR and PD-L1 on NK cell recruitment. The results of our study demonstrated that A2BR and PD-L1 are co-expressed in OSCC. Moreover, treatment with BAY60-6583 induced PD-L1 expression in the CAL-27 cells, which was partially reduced in cells pretreated with PDTC, suggesting that A2BR agonists induce PD-L1 expression via the induction of the NF-κ B signaling pathway. Furthermore, high A2BR expression in OSCC was associated with lower infiltration of NK cells. Additionally, our results demonstrated that treatment with MRS-1706 (an A2BR inverse agonist) and/or CD274 (a PD-L1-neutralizing antibody) promoted NK cell recruitment and cytotoxicity against OSCC cells. Altogether, our findings highlight the synergistic effect of co-inhibition of A2BR and PD-L1 in the treatment of OSCC via the modulation of NK cell recruitment and cytotoxicity.

Glutathione-Mediated Neuroprotective Effect of Purine Derivatives.

Numerous basic studies have reported on the neuroprotective properties of several purine derivatives such as caffeine and uric acid (UA). Epidemiological studies have also shown the inverse association of appropriate caffeine intake or serum urate levels with neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson's disease (PD). The well-established neuroprotective mechanisms of caffeine and UA involve adenosine A2A receptor antagonism and antioxidant activity, respectively. Our recent study found that another purine derivative, paraxanthine, has neuroprotective effects similar to those of caffeine and UA. These purine derivatives can promote neuronal cysteine uptake through excitatory amino acid carrier protein 1 (EAAC1) to increase neuronal glutathione (GSH) levels in the brain. This review summarizes the GSH-mediated neuroprotective effects of purine derivatives. Considering the fact that GSH depletion is a manifestation in the brains of AD and PD patients, administration of purine derivatives may be a new therapeutic approach to prevent or delay the onset of these neurodegenerative diseases.

Xanthine-Dopamine Hybrid Molecules as Multitarget Drugs with Potential for the Treatment of Neurodegenerative Diseases.

Multitarget drugs based on a hybrid dopamine-xanthine core were designed as potential drug candidates for the treatment of neurodegenerative diseases. Monoamine oxidase B (MAO-B) inhibitors with significant ancillary A2A adenosine receptor (A2AAR) antagonistic properties were further developed to exhibit additional phosphodiesterase-4 and -10 (PDE4/10) inhibition and/or dopamine D2 receptor (D2R) agonistic activity. While all of the designed compounds showed MAO-B inhibition in the nanomolar range mostly combined with submicromolar A2AAR affinity, significant enhancement of PDE-inhibitory and D2R-agonistic activity was additionally reached for some compounds through various structural modifications. The final multitarget drugs also showed promising antioxidant properties in vitro. In order to evaluate their potential neuroprotective effect, representative ligands were tested in a cellular model of toxin-induced neurotoxicity. As a result, protective effects against oxidative stress in neuroblastoma cells were observed, confirming the utility of the applied strategy. Further evaluation of the newly developed multitarget ligands in preclinical models of Alzheimer's and Parkinson's diseases is warranted.

A2BAR Antagonism Decreases the Glomerular Expression and Secretion of Chemoattractants for Monocytes and the Pro-Fibrotic M2 Macrophages Polarization during Diabetic Nephropathy.

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

Structure-based virtual screening discovers potent and selective adenosine A1 receptor antagonists.

Development of subtype-selective leads is essential in drug discovery campaigns targeting G protein-coupled receptors (GPCRs). Herein, a structure-based virtual screening approach to rationally design subtype-selective ligands was applied to the A1 and A2A adenosine receptors (A1R and A2AR). Crystal structures of these closely related subtypes revealed a non-conserved subpocket in the binding sites that could be exploited to identify A1R selective ligands. A library of 4.6 million compounds was screened computationally against both receptors using molecular docking and 20 A1R selective ligands were predicted. Of these, seven antagonized the A1R with micromolar activities and several compounds displayed slight selectivity for this subtype. Twenty-seven analogs of two discovered scaffolds were designed, resulting in antagonists with nanomolar potency and up to 76-fold A1R-selectivity. Our results show the potential of structure-based virtual screening to guide discovery and optimization of subtype-selective ligands, which could facilitate the development of safer drugs.

Dual A1 and A2A adenosine receptor antagonists, methoxy substituted 2-benzylidene-1-indanone, suppresses intestinal postprandial glucose and attenuates hyperglycaemia in fructose-streptozotocin diabetic rats.

BACKGROUND/AIM: Recent research suggests that adenosine receptors (ARs) influence many of the metabolic abnormalities associated with diabetes. A non-xanthine benzylidene indanone derivative 2-(3,4-dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1 H-inden-1-one (2-BI), has shown to exhibit higher affinity at A1/A2A ARs compared to caffeine. Due to its structural similarity to caffeine, and the established antidiabetic effects of caffeine, the current study was initiated to explore the possible antidiabetic effect of 2-BI. METHODS: The study was designed to assess the antidiabetic effects of several A1 and/or A2A AR antagonists, via intestinal glucose absorption and glucose-lowering effects in fructose-streptozotocin (STZ) induced diabetic rats. Six-week-old male Sprague-Dawley rats were induced with diabetes via fructose and streptozotocin. Rats were treated for 4 weeks with AR antagonists, metformin and pioglitazone, respectively. Non-fasting blood glucose (NFBG) was determined weekly and the oral glucose tolerance test (OGTT) was conducted at the end of the intervention period. RESULTS: Dual A1/A2A AR antagonists (caffeine and 2-BI) decreased glucose absorption in the intestinal membrane significantly (p < 0.01), while the selective A2A AR antagonist (Istradefylline), showed the highest significant (p < 0.001) reduction in intestinal glucose absorption. The selective A1 antagonist (DPCPX) had the least significant (p < 0.05) reduction in glucose absorption. Similarly, dual A1/A2A AR antagonists and selective A2A AR antagonists significantly reduced non-fast blood glucose and improved glucose tolerance in diabetic rats from the first week of the treatment. Conversely, the selective A1 AR antagonist did not reduce non-fast blood glucose significantly until the 4th week of treatment. 2-BI, caffeine and istradefylline compared well with standard antidiabetic treatments, metformin and pioglitazone, and in some cases performed even better. CONCLUSION: 2-BI exhibited good antidiabetic activity by reducing intestinal postprandial glucose absorption and improving glucose tolerance in a diabetic animal model. The dual antagonism of A1/A2A ARs presents a positive synergism that could provide a new possibility for the treatment of diabetes.

Selective Deuteration Improves the Affinity of Adenosine A2A Receptor Ligands: A Computational Case Study with Istradefylline and Caffeine.

We used a range of computational techniques to assess the effect of selective C-H deuteration on the antagonist istradefylline affinity for the adenosine A2A receptor, which was discussed relative to its structural analogue caffeine, a well-known and likely the most widely used stimulant. The obtained results revealed that smaller caffeine shows high receptor flexibility and exchanges between two distinct poses, which agrees with crystallographic data. In contrast, the additional C8-trans-styryl fragment in istradefylline locks the ligand within a uniform binding pose, while contributing to the affinity through the C-H···π and π···π contacts with surface residues, which, together with its much lower hydration prior to binding, enhances the affinity over caffeine. In addition, the aromatic C8-unit shows a higher deuteration sensitivity over the xanthine part, so when both of its methoxy groups are d6-deuterated, the affinity improvement is -0.4 kcal mol-1, which surpasses the overall affinity gain of -0.3 kcal mol-1 in the perdeuterated d9-caffeine. Yet, the latter predicts around 1.7-fold potency increase, being relevant for its pharmaceutical implementations, and also those within the coffee and energy drink production industries. Still, the full potential of our strategy is achieved in polydeuterated d19-istradefylline, whose A2A affinity improves by -0.6 kcal mol-1, signifying a 2.8-fold potency increase that strongly promotes it as a potential synthetic target. This knowledge supports deuterium application in drug design, and while the literature already reports about over 20 deuterated drugs currently in the clinical development, it is easily foreseen that more examples will hit the market in the years to come. With this in mind, we propose that the devised computational methodology, involving the ONIOM division of the QM region for the ligand and the MM region for its environment, with an implicit quantization of nuclear motions relevant for the H/D exchange, allows fast and efficient estimates of the binding isotope effects in any biological system.

Discovery of Pyridinone Derivatives as Potent, Selective, and Orally Bioavailable Adenosine A2A Receptor Antagonists for Cancer Immunotherapy.

Recent studies and clinical evidence have strongly supported the development of adenosine A2A receptor (A2AR) antagonists as novel approaches for cancer immunotherapy. By screening our in-house compound library, a pyridinone hit compound (1) with weak A2AR antagonistic activity was identified. Further structure-activity relationship studies revealed a series of pyridinone derivatives with strong potency. Compound 38 stood out with a potent A2AR antagonistic activity (IC50 = 29.0 nM), good mouse liver microsomal metabolic stability (t1/2 = 86.1 min), and excellent oral bioavailability (F = 86.1%). Of note, 38 effectively enhanced the activation and killing ability of T cells in vitro by down-regulation of immunosuppressive molecules (LAG-3 and TIM-3) and up-regulation of effector molecules (GZMB, IFNG, and IL-2). Moreover, 38 exhibited excellent in vivo antitumor activity with a tumor growth inhibition (TGI) of 56.0% in the MC38 tumor model via oral administration, demonstrating its potential as a novel A2AR antagonist candidate for cancer immunotherapy.

Role of adenosine A2A receptors in hot and cold cognition: Effects of single-dose istradefylline in healthy volunteers.

The role of the adenosine neurochemical system in human cognition is under-studied, despite such receptors being distributed throughout the brain. The aim of this study was to shed light on the role of the adenosine A2A receptors in human cognition using single-dose istradefylline. Twenty healthy male participants, aged 19-49, received 20 mg istradefylline and placebo, in a randomized, double-blind, placebo-controlled cross-over design. Cognition was assessed using computerized cognitive tests, covering both cold (non-emotional) and hot (emotion-laden) domains. Cardiovascular data were recorded serially. Cognitive effects of istradefylline were explored using repeated measures analysis of variance and paired t-tests as appropriate. On the EMOTICOM battery, there was a significant effect of istradefylline versus placebo on the Social Information Preference task (t = 2.50, p = 0.02, d=-0.59), indicating that subjects on istradefylline interpreted social situations more positively. No other significant effects were observed on other cognitive tasks, nor in terms of cardiovascular measures (pulse and blood pressure). De-briefing indicated that blinding was successful, both for participants and the research team. Further exploration of the role of adenosine A2A receptors in emotional processing may be valuable, given that abnormalities in related cognitive functions are implicated in neuropsychiatric disorders. The role of adenosine systems in human cognition requires further clarification, including with different doses of istradefylline and over different schedules of administration.

In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A2A Receptor Antagonist/Inverse Agonist.

KW-6356 is a novel adenosine A2A (A2A) receptor antagonist/inverse agonist, and its efficacy as monotherapy in Parkinson's disease (PD) patients has been reported. Istradefylline is a first-generation A2A receptor antagonist approved for use as adjunct treatment to levodopa/decarboxylase inhibitor in adult PD patients experiencing "OFF" episodes. In this study, we investigated the in vitro pharmacological profile of KW-6356 as an A2A receptor antagonist/inverse agonist and the mode of antagonism and compared them with istradefylline. In addition, we determined cocrystal structures of A2A receptor in complex with KW-6356 and istradefylline to explore the structural basis of the antagonistic properties of KW-6356. Pharmacological studies have shown that KW-6356 is a potent and selective ligand for the A2A receptor (the -log of inhibition constant = 9.93 ± 0.01 for human receptor) with a very low dissociation rate from the receptor (the dissociation kinetic rate constant = 0.016 ± 0.006 minute-1 for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, whereas istradefylline exhibits surmountable antagonism. Crystallography of KW-6356- and istradefylline-bound A2A receptor have indicated that interactions with His2506.52 and Trp2466.48 are essential for the inverse agonism, whereas the interactions at both deep inside the orthosteric pocket and the pocket lid stabilizing the extracellular loop conformation may contribute to the insurmountable antagonism of KW-6356. These profiles may reflect important differences in vivo and help predict better clinical performance. SIGNIFICANCE STATEMENT: KW-6356 is a potent and selective adenosine A2A receptor antagonist/inverse agonist and exhibits insurmountable antagonism, whereas istradefylline, a first-generation adenosine A2A receptor antagonist, exhibits surmountable antagonism. Structural studies of adenosine A2A receptor in complex with KW-6356 and istradefylline explain the characteristic differences in the pharmacological properties of KW-6356 and istradefylline.

Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes.

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A2A and A3ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A2AAR were Ki 144-316 nM for 10, 12, and 19, and at A3AR affinity of Ki 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A2AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A3AR characterization in whole cells by flow cytometry (Kd 11.8 nM), and its bitopic interaction mode with an A3AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A2AAR, ~ 50-fold vs. A1AR and A2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A3AR.

Exploring the Effect of Halogenation in a Series of Potent and Selective A2B Adenosine Receptor Antagonists.

The modulation of the A2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A2BAR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (di- and mono-)halogenation at positions 7 and/or 8 on both A2BAR affinity and pharmacokinetic properties of a collection of A2BAR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A2BAR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogen-size-dependent decay in affinity. Eight novel A2BAR ligands exhibited remarkable affinity (Ki < 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells.

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.

The role of adenosine in alcohol-induced respiratory suppression.

Alcohol-related poisoning is the foremost cause of death resulting from excessive acute alcohol consumption. Respiratory failure is crucial to the pathophysiology of fatal alcohol poisoning. Alcohol increases accumulation of extracellular adenosine. Adenosine suppresses breathing. The goal of this investigation was to test the hypothesis that adenosine signaling contributes to alcohol-induced respiratory suppression. In the first experiment, the breathing of mice was monitored following an injection of the non-selective adenosine receptor antagonist caffeine (40 mg/kg), alcohol (5 g/kg), or alcohol and caffeine combined. Caffeine reduced alcohol-induced respiratory suppression suggesting that adenosine contributes to the effects of alcohol on breathing. The second experiment utilized the same experimental design, but with the blood brain barrier impermeant non-selective adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT, 60 mg/kg) instead of caffeine. 8-SPT did not reduce alcohol-induced respiratory suppression suggesting that adenosine is contributing to alcohol-induced respiratory suppression in the central nervous system. The third and fourth experiments used the same experimental design as the first, but with the selective A1 receptor antagonist DPCPX (1 mg/kg) and the selective A2A receptor antagonist istradefylline (3.3 mg/kg). Istradefylline, but not DPCPX, reduced alcohol-induced respiratory suppression indicating an A2A receptor mediated effect. In the fifth experiment, alcohol-induced respiratory suppression was evaluated in Adk+/- mice which have impaired adenosine metabolism. Alcohol-induced respiratory suppression was exacerbated in Adk+/- mice. These findings indicate that adenosinergic signaling contributes to alcohol-induced respiratory suppression. Improving our understanding of how alcohol affects breathing may lead to better treatment strategies and better outcomes for patients with severe alcohol poisoning.

Preliminary Evidence of the Potent and Selective Adenosine A2B Receptor Antagonist PSB-603 in Reducing Obesity and Some of Its Associated Metabolic Disorders in Mice.

The adenosine A2A and A2B receptors are promising therapeutic targets in the treatment of obesity and diabetes since the agonists and antagonists of these receptors have the potential to positively affect metabolic disorders. The present study investigated the link between body weight reduction, glucose homeostasis, and anti-inflammatory activity induced by a highly potent and specific adenosine A2B receptor antagonist, compound PSB-603. Mice were fed a high-fat diet for 14 weeks, and after 12 weeks, they were treated for 14 days intraperitoneally with the test compound. The A1/A2A/A2B receptor antagonist theophylline was used as a reference. Following two weeks of treatment, different biochemical parameters were determined, including total cholesterol, triglycerides, glucose, TNF-α, and IL-6 blood levels, as well as glucose and insulin tolerance. To avoid false positive results, mouse locomotor and spontaneous activities were assessed. Both theophylline and PSB-603 significantly reduced body weight in obese mice. Both compounds had no effects on glucose levels in the obese state; however, PSB-603, contrary to theophylline, significantly reduced triglycerides and total cholesterol blood levels. Thus, our observations showed that selective A2B adenosine receptor blockade has a more favourable effect on the lipid profile than nonselective inhibition.

The adenosine A2A receptor is a therapeutic target in neurological, heart and oncogenic diseases.

INTRODUCTION: Following approval of antagonists in Parkinson's disease therapy, the A2A adenosine receptor (A2AR) is gaining interest as a target to combat a variety of diseases. AREAS COVERED: This review focuses on the therapeutic potential of targeting A2AR inside but also outside the central nervous system, more precisely to combat cardiac arrhythmias and to boost immune-based cancer therapies. The mechanism of regulation of the immune system by adenosine (Ado) is complex since several actors are involved, namely the enzymes that produce and degrade the compound and the four Ado receptors. Antagonists of A2A or andadenosine A2B Ado receptors lead to reduce their activation intracellular cAMP levels thus boosting immune responses. The literature search methodology consisted of reviewing the authors own collection of papers plus searches in the PubMed and Google Scholar databases from March 2021 to August 2022. EXPERT OPINION: There is a pending issue, namely, how to demonstrate the neuroprotective potential of A2AR antagonists that would lead to delay neurodegenerative disease progression. In addition, A2AR antagonists and, eventually, dual A2A/A2B receptor antagonists have potential in the treatment of heart arrythmias and cancer. If adequate resources are allocated, it is a matter of time to confirm whether or not these possibilities become a reality.

Adenosine A2a Receptor Antagonism Restores Additive Cytotoxicity by Cytotoxic T Cells in Metabolically Perturbed Tumors.

Cytotoxic T lymphocytes (CTL) are antigen-specific effector cells with the ability to eradicate cancer cells in a contact-dependent manner. Metabolic perturbation compromises the CTL effector response in tumor subregions, resulting in failed cancer cell elimination despite the infiltration of tumor-specific CTLs. Restoring the functionality of these tumor-infiltrating CTLs is key to improve immunotherapy. Extracellular adenosine is an immunosuppressive metabolite produced within the tumor microenvironment. Here, by applying single-cell reporter strategies in 3D collagen cocultures in vitro and progressing tumors in vivo, we show that adenosine weakens one-to-one pairing of activated effector CTLs with target cells, thereby dampening serial cytotoxic hit delivery and cumulative death induction. Adenosine also severely compromised CTL effector restimulation and expansion. Antagonization of adenosine A2a receptor (ADORA2a) signaling stabilized and prolonged CTL-target cell conjugation and accelerated lethal hit delivery by both individual contacts and CTL swarms. Because adenosine signaling is a near-constitutive confounding parameter in metabolically perturbed tumors, ADORA2a targeting represents an orthogonal adjuvant strategy to enhance immunotherapy efficacy.

In silico studies of natural product-like caffeine derivatives as potential MAO-B inhibitors/AA2AR antagonists for the treatment of Parkinson's disease.

Parkinson's disease is considered the second most frequent neurodegenerative disease. It is described by the loss of dopaminergic neurons in the mid-brain. For many decades, L-DOPA has been considered as the gold standard for treating Parkinson's disease motor symptoms, however, due to the decrease of efficacy, in the long run, there is an urgent need for novel antiparkinsonian drugs. Caffeine derivatives have been reported several times for their neuroprotective properties and dual blockade of monoamine oxidase (MAO) and adenosine A2A receptors (AA2AR). Natural products are currently attracting more focus due to structural diversity and safety in contrast to synthetic drugs. In the present work, computational studies were conducted on natural product-like caffeine derivatives to search for novel potent candidates acting as dual MAO-B inhibitors/AA2AR antagonists for Parkinson's disease. Our findings revealed two natural products among the top hits: CNP0202316 and CNP0365210 fulfill the requirements of drugs acting on the brain. The selected lead compounds were further studied using molecular dynamics simulation to assess their stability with MAO-B. Current findings might shift the interest towards natural-based compounds and could be exploited to further optimize caffeine derivatives into a successful dual-target-directed drug for managing and halting the neuronal damage in Parkinson's disease patients.