Interaction of Purine and its Derivatives with A1, A2-Adenosine Receptors and Vascular Endothelial Growth Factor Receptor-1 (Vegf-R1) as a Therapeutic Alternative to Treat Cancer.

BACKGROUND: There are several studies that indicate that cancer development may be conditioned by the activation of some biological systems that involve the interaction of different biomolecules, such as adenosine and vascular endothelial growth factor. These biomolecules have been targeted of some drugs for treat of cancer; however, there is little information on the interaction of purine derivatives with adenosine and vascular endothelial growth factor receptor (VEGF-R1). OBJECTIVE: The aim of this research was to determine the possible interaction of purine (1: ) and their derivatives (2-31: ) with A1, A2-adenosine receptors, and VEGF-R1. METHODS: Theoretical interaction of purine and their derivatives with A1, A2-adenosine receptors and VEGF-R1 was carried out using the 5uen, 5mzj and 3hng proteins as theoretical tools. Besides, adenosine, cgs-15943, rolofylline, cvt-124, wrc-0571, luf-5834, cvt-6883, AZD-4635, cabozantinib, pazopanib, regorafenib, and sorafenib drugs were used as controls. RESULTS: The results showed differences in the number of aminoacid residues involved in the interaction of purine and their derivatives with 5uen, 5mzj and 3hng proteins compared with the controls. Besides, the inhibition constants (Ki) values for purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: were lower compared with the controls CONCLUSIONS: Theoretical data suggest that purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: could produce changes in cancer cell growth through inhibition of A1, A2-adenosine receptors and VEGFR-1 inhibition. These data indicate that these purine derivatives could be a therapeutic alternative to treat some types of cancer.

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.

The ADORA1 mutation linked to early-onset Parkinson's disease alters adenosine A1-A2A receptor heteromer formation and function.

Adenosine modulates neurotransmission through inhibitory adenosine A1 receptors (A1Rs) and stimulatory A2A receptors (A2ARs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson's disease (PD). An autosomal-recessive mutation (G2797.44S) within the transmembrane helix (TM) 7 of A1R (A1RG279S) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A1R and the A1R-A2AR heteromer. Our results revealed that the G2797.44S mutation does not alter A1R expression, ligand binding, constitutive activity or coupling to transducer proteins (Gαi, Gαq, Gα12/13, Gαs, ß-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A1RG279S weakened the ability of A1R to heteromerize with A2AR, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A1R imposes on the constitutive activity and agonist-induced activation of the A2AR. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G2797.44S mutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A2AR not forming heteromers with A1R could represent a main pathogenetic mechanism of the EOPD associated with the G2797.44S ADORA1 mutation.

A2A Adenosine Receptor: A Possible Therapeutic Target for Alzheimer's Disease by Regulating NLRP3 Inflammasome Activity?

The A2A adenosine receptor, a member of the P1 purinergic receptor family, plays a crucial role in the pathophysiology of different neurodegenerative illnesses, including Alzheimer's disease (AD). It regulates both neurons and glial cells, thus modulating synaptic transmission and neuroinflammation. AD is a complex, progressive neurological condition that is the leading cause of dementia in the world's old population (>65 years of age). Amyloid peptide-ß extracellular accumulation and neurofibrillary tangles constitute the principal etiologic tracts, resulting in apoptosis, brain shrinkage, and neuroinflammation. Interestingly, a growing body of evidence suggests a role of NLRP3 inflammasome as a target to treat neurodegenerative diseases. It represents a tripartite multiprotein complex including NLRP3, ASC, and procaspase-1. Its activation requires two steps that lead with IL-1ß and IL-18 release through caspase-1 activation. NLRP3 inhibition provides neuroprotection, and in recent years adenosine, through the A2A receptor, has been reported to modulate NLRP3 functions to reduce organ damage. In this review, we describe the role of NLRP3 in AD pathogenesis, both alone and in connection to A2A receptor regulation, in order to highlight a novel approach to address treatment of AD.

Antioxidant and Antiinflammatory Effects of Epilobium parviflorum, Melilotus officinalis and Cardiospermum halicacabum Plant Extracts in Macrophage and Microglial Cells.

BACKGROUND: We investigated the phenolic content characterizing different plant extracts from Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis, their antioxidant, antiinflammatory effects, and their mechanism of action. METHODS: plant samples were macerated in 40% ethanol or hot/ cold glycerate and assessed for polyphenols content. The antioxidant activity was investigated by DPPH radical scavenging assay and H2DCFDA test in LPS-stimulated RAW264.7 macrophages and N9 microglial cells. MTS experiments and antiinflammatory properties verified cellular toxicity through NO assay. Interaction with A2A adenosine receptors was evaluated through binding assays using [3H]ZM241385 radioligand. RESULTS: Polyphenols were present in 40% ethanol plant extract, which at 0.1-10 µg/µL achieved good antioxidant effects, with a DPPH radical scavenging rate of about 90%. In LPS-stimulated cells, these plant extracts, at 1µg/µL, did not affect cell vitality, displayed significant inhibition of H2DCFDA and NO production, and inhibited ZM 241385 binding in CHO cells transfected with A2A receptors. RAW 264.7 and N9 cells presented a density of them quantified in 60 ± 9 and 45 ± 5 fmol/mg of protein, respectively. CONCLUSION: Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis extracts may be considered a source of agents for treating disorders related to oxidative stress and inflammation.

Alcohol inhibits alveolar fluid clearance through the epithelial sodium channel via the A2 adenosine receptor in acute lung injury.

Chronic alcohol abuse increases the risk of mortality and poor outcomes in patients with acute respiratory distress syndrome. However, the underlying mechanisms remain to be elucidated. The present study aimed to investigate the effects of chronic alcohol consumption on lung injury and clarify the signaling pathways involved in the inhibition of alveolar fluid clearance (AFC). In order to produce rodent models with chronic alcohol consumption, wild­type C57BL/6 mice were treated with alcohol. A2a adenosine receptor (AR) small interfering (si)RNA or A2bAR siRNA were transfected into the lung tissue of mice and primary rat alveolar type II (ATII) cells. The rate of AFC in lung tissue was measured during exposure to lipopolysaccharide (LPS). Epithelial sodium channel (ENaC) expression was determined to investigate the mechanisms underlying alcohol­induced regulation of AFC. In the present study, exposure to alcohol reduced AFC, exacerbated pulmonary edema and worsened LPS­induced lung injury. Alcohol caused a decrease in cyclic adenosine monophosphate (cAMP) levels and inhibited α­ENaC, ß­ENaC and γ­ENaC expression levels in the lung tissue of mice and ATII cells. Furthermore, alcohol decreased α­ENaC, ß­ENaC and γ­ENaC expression levels via the A2aAR or A2bAR­cAMP signaling pathways in vitro. In conclusion, the results of the present study demonstrated that chronic alcohol consumption worsened lung injury by aggravating pulmonary edema and impairing AFC. An alcohol­induced decrease of α­ENaC, ß­ENaC and γ­ENaC expression levels by the A2AR­mediated cAMP pathway may be responsible for the exacerbated effects of chronic alcohol consumption in lung injury.

Modulation of Blood-Brain Barrier Permeability by Activating Adenosine A2 Receptors in Oncological Treatment.

The blood-brain barrier (BBB) plays an important protective role in the central nervous system and maintains its homeostasis. It regulates transport into brain tissue and protects neurons against the toxic effects of substances circulating in the blood. However, in the case of neurological diseases or primary brain tumors, i.e., gliomas, the higher permeability of the blood-derived substances in the brain tissue is necessary. Currently applied methods of treatment for the primary brain neoplasms include surgical removal of the tumor, radiation therapy, and chemotherapy. Despite the abovementioned treatment methods, the prognosis of primary brain tumors remains bad. Moreover, chemotherapy options seem to be limited due to low drug penetration into the cancerous tissue. Modulation of the blood-brain barrier permeability may contribute to an increase in the concentration of the drug in the CNS and thus increase the effectiveness of therapy. Interestingly, endothelial cells in cerebral vessels are characterized by the presence of adenosine 2A receptors (A2AR). It has been shown that substances affecting these receptors regulate the permeability of the BBB. The mechanism of increasing the BBB permeability by A2AR agonists is the actin-cytoskeletal reorganization and acting on the tight junctions. In this case, the A2AR seems to be a promising therapy target. This article aims to assess the possibility of increasing the BBB permeability through A2AR agonists to increase the effectiveness of chemotherapy and to improve the results of cancer therapy.

Combined Therapy of A1AR Agonists and A2AAR Antagonists in Neuroinflammation.

Alzheimer's, Parkinson's, and multiple sclerosis are neurodegenerative diseases related by neuronal degeneration and death in specific areas of the central nervous system. These pathologies are associated with neuroinflammation, which is involved in disease progression, and halting this process represents a potential therapeutic strategy. Evidence suggests that microglia function is regulated by A1 and A2A adenosine receptors (AR), which are considered as neuroprotective and neurodegenerative receptors, respectively. The manuscript's aim is to elucidate the role of these receptors in neuroinflammation modulation through potent and selective A1AR agonists (N6-cyclopentyl-2'- or 3'-deoxyadenosine substituted or unsubstituted in 2 position) and A2AAR antagonists (9-ethyl-adenine substituted in 8 and/or in 2 position), synthesized in house, using N13 microglial cells. In addition, the combined therapy of A1AR agonists and A2AAR antagonists to modulate neuroinflammation was evaluated. Results showed that A1AR agonists were able, to varying degrees, to prevent the inflammatory effect induced by cytokine cocktail (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and interferon (IFN)-γ), while A2AAR antagonists showed a good ability to counteract neuroinflammation. Moreover, the effect achieved by combining the two most effective compounds (1 and 6) in doses previously found to be non-effective was greater than the treatment effect of each of the two compounds used separately at maximal dose.

A2B Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination.

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing A2BR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, A2BRs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by A2BRs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the A2BRs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.

Purinergic Signaling in Pancreas-From Physiology to Therapeutic Strategies in Pancreatic Cancer.

The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y2 and P2Y12 receptors, as well as A2 receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.

Isoprenoid-chained lipid EROCOC17+4: a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography.

In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to - 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method.

CHARMM-GUI Free Energy Calculator for Absolute and Relative Ligand Solvation and Binding Free Energy Simulations.

Alchemical free energy simulations have long been utilized to predict free energy changes for binding affinity and solubility of small molecules. However, while the theoretical foundation of these methods is well established, seamlessly handling many of the practical aspects regarding the preparation of the different thermodynamic end states of complex molecular systems and the numerous processing scripts often remains a burden for successful applications. In this work, we present CHARMM-GUI Free Energy Calculator (http://www.charmm-gui.org/input/fec) that provides various alchemical free energy perturbation molecular dynamics (FEP/MD) systems with input and post-processing scripts for NAMD and GENESIS. Four submodules are available: Absolute Ligand Binder (for absolute ligand binding FEP/MD), Relative Ligand Binder (for relative ligand binding FEP/MD), Absolute Ligand Solvator (for absolute ligand solvation FEP/MD), and Relative Ligand Solvator (for relative ligand solvation FEP/MD). Each module is designed to build multiple systems of a set of selected ligands at once for high-throughput FEP/MD simulations. The capability of Free Energy Calculator is illustrated by absolute and relative solvation FEP/MD of a set of ligands and absolute and relative binding FEP/MD of a set of ligands for T4-lysozyme in solution and the adenosine A2A receptor in a membrane. The calculated free energy values are overall consistent with the experimental and published free energy results (within ∼1 kcal/mol). We hope that Free Energy Calculator is useful to carry out high-throughput FEP/MD simulations in the field of biomolecular sciences and drug discovery.

Interplay of the adenosine system and NO in control of renal haemodynamics and excretion: Comparison of normoglycaemic and streptozotocin diabetic rats.

The adenosine (Ado) system may participate in regulation of kidney function in diabetes mellitus (DM), therefore we explored its role and interrelation with NO in the control of renal circulation and excretion in normoglycemic (NG) and streptozotocin-diabetic (DM) rats. Effects of theophylline (Theo), a non-selective Ado receptor antagonist, were examined in anaesthetized NG or in streptozotocin induced diabetic (DM) rats, untreated or after blockade of NO synthesis with l-NAME. We measured arterial blood pressure (MABP), whole kidney blood flow and renal regional flows: cortical and outer- and inner-medullary (IMBF), determined as laser-Doppler fluxes. Renal excretion of water, total solutes and sodium and in situ renal tissue NO signal (selective electrodes) were also determined. Theo experiments disclosed minor baseline vasoconstrictor and vasodilator tone in the kidney of NG and DM rats, respectively. NO blockade increased baseline MABP and decreased renal haemodynamics, similarly in NG and DM rats, indicating comparable vasodilator influence of NO in the two groups. Unexpectedly, in all rats with intact NO synthesis, Ado receptor blockade increased kidney tissue NO. In NO-deficient NG and DM rats, Ado receptor blockade induced comparable renal vasodilatation, suggesting similar vasoconstrictor influence of the Ado system. However, DM rats showed an unexplained association of decreased MABP and IMBF and increased NO signal. Higher baseline renal excretion in DM rats indicated inhibition of renal tubular reabsorption due to the prevalence of natriuretic A2 over antinatriuretic A1 receptors. In conclusion, the experiments provided new insights in functional interrelation of adenosine and NO in normoglycaemia and streptozotocin-diabetes.

Single exercise stress reduces central neurotrophins levels and adenosine A1 and A2 receptors expression, but does not revert opioid-induced hyperalgesia in rats.

BACKGROUND: This study assessed the effects of an acute stress model upon the long-term hyperalgesia induced by repeated morphine administration in neonatal rats. We also evaluated neurotrophins and cytokines levels; expressions of adenosine and acetylcholine receptors, and acetylcholinesterase enzyme at the spinal cord. MATERIAL AND METHODS: Male Wistar rats were subjected to morphine or saline administration from P8 to P14. Thermal hyperalgesia and mechanical hyperesthesia were assessed using the hot plate (HP) and von Frey (vF) tests, respectively, at postnatal day P30 and P60. After baseline measurements, rats were subjected to a single exercise session, as an acute stress model, at P30 or P60. We measured the levels of BDNF and NGF, interleukin-6, and IL-10 in the cerebral cortex and the brainstem; and the expression levels of adenosine and muscarinic receptors, as well as acetylcholinesterase (AChE) enzyme at the spinal cord. RESULTS: A stress exercise session was not able to revert the morphine-induced hyperalgesia. The morphine and exercise association in rats induced a decrease in the neurotrophins brainstem levels, and A1 , A2A , A2B receptors expression in the spinal cord, and an increase in the IL-6 cortical levels. The exercise reduced M2 receptors expression in the spinal cord of naive rats, while morphine prevented this effect. CONCLUSIONS: Single session of exercise does not revert hyperalgesia induced by morphine in rats; however, morphine plus exercise modulate neurotrophins, IL-6 central levels, and expression of adenosine receptors.

Adenosine and Metabotropic Glutamate Receptors Are Present in Blood Serum and Exosomes from SAMP8 Mice: Modulation by Aging and Resveratrol.

Adenosine (ARs) and metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) that are modulated in the brain of SAMP8 mice, an animal model of Alzheimer's disease (AD). In the present work, it is shown the presence of ARs and mGluRs in blood serum and derived exosomes from SAMP8 mice as well as its possible modulation by aging and resveratrol (RSV) consumption. In blood serum, adenosine A1 and A2A receptors remained unaltered from 5 to 7 months of age. However, an age-related decrease in adenosine level was observed, while 5'-Nucleotidase activity was not modulated. Regarding the glutamatergic system, it was observed a decrease in mGluR5 density and glutamate levels in older mice. In addition, dietary RSV supplementation caused an age-dependent modulation in both adenosinergic and glutamatergic systems. These GPCRs were also found in blood serum-derived exosomes, which might suggest that these receptors could be released into circulation via exosomes. Interestingly, changes elicited by age and RSV supplementation on mGluR5 density, and adenosine and glutamate levels were similar to that detected in whole-brain. Therefore, we might suggest that the quantification of these receptors, and their corresponding endogenous ligands, in blood serum could have predictive value for early diagnosis in combination with other distinctive hallmarks of AD.

The Detrimental Action of Adenosine on Glutamate-Induced Cytotoxicity in PC12 Cells Can Be Shifted towards a Neuroprotective Role through A1AR Positive Allosteric Modulation.

Glutamate cytotoxicity is implicated in neuronal death in different neurological disorders including stroke, traumatic brain injury, and neurodegenerative diseases. Adenosine is a nucleoside that plays an important role in modulating neuronal activity and its receptors have been identified as promising therapeutic targets for glutamate cytotoxicity. The purpose of this study is to elucidate the role of adenosine and its receptors on glutamate-induced injury in PC12 cells and to verify the protective effect of the novel A1 adenosine receptor positive allosteric modulator, TRR469. Flow cytometry experiments to detect apoptosis revealed that adenosine has a dual role in glutamate cytotoxicity, with A2A and A2B adenosine receptor (AR) activation exacerbating and A1 AR activation improving glutamate-induced cell injury. The overall effect of endogenous adenosine in PC12 cells resulted in a facilitating action on glutamate cytotoxicity, as demonstrated by the use of adenosine deaminase and selective antagonists. However, enhancing the action of endogenous adenosine on A1ARs by TRR469 completely abrogated glutamate-mediated cell death, caspase 3/7 activation, ROS production, and mitochondrial membrane potential loss. Our results indicate a novel potential therapeutic strategy against glutamate cytotoxicity based on the positive allosteric modulation of A1ARs.

Synthesis and Structure Activity Relationships of Chalcone based Benzocycloalkanone Derivatives as Adenosine A1 and/or A2A Receptor Antagonists.

Adenosine A1 and/or A2A receptor antagonists hold promise for the potential treatment of neurological conditions, such as Parkinson's disease. Herein, a total of seventeen benzocycloalkanone derivatives were synthesised and evaluated for affinity towards adenosine receptors (A1 and A2A AR). The obtained results allowed for the conclusion that affinity and/or selectivity of the 2-benzylidene-1-indanone and -tetralone derivatives toward A1 and/or A2A ARs may be modulated by the nature of the substituents (either -OH, -OCH3 or morpholine) attached at position C4 of the 1-indanone core and C5 of the 1-tetralone core as well as the meta (C3') and/or para (C4') position(s) on ring B. Several compounds (2A: -B: , 3B: -C: and 4A: -B: ) possessed affinity for the A1 and/or A2A AR below 10 µM. Additionally, compounds 2A: , 3B: and 4A: were A1 AR antagonists. These results, once again, confirmed the importance of C4 methoxy-group substitution on ring A in combination with meta (C3') and/or para (C4') hydroxyl-group substitution ring B of the 2-benzylidene-1-indanone scaffold leading to drug-like compounds 1H: and 1J: with affinity in the nanomolar-range.

Identification of Novel Chemical Entities for Adenosine Receptor Type 2A Using Molecular Modeling Approaches.

Adenosine Receptor Type 2A (A2AAR) plays a role in important processes, such as anti-inflammatory ones. In this way, the present work aimed to search for compounds by pharmacophore-based virtual screening. The pharmacokinetic/toxicological profiles of the compounds, as well as a robust QSAR, predicted the binding modes via molecular docking. Finally, we used molecular dynamics to investigate the stability of interactions from ligand-A2AAR. For the search for A2AAR agonists, the UK-432097 and a set of 20 compounds available in the BindingDB database were studied. These compounds were used to generate pharmacophore models. Molecular properties were used for construction of the QSAR model by multiple linear regression for the prediction of biological activity. The best pharmacophore model was used by searching for commercial compounds in databases and the resulting compounds from the pharmacophore-based virtual screening were applied to the QSAR. Two compounds had promising activity due to their satisfactory pharmacokinetic/toxicological profiles and predictions via QSAR (Diverset 10002403 pEC50 = 7.54407; ZINC04257548 pEC50 = 7.38310). Moreover, they had satisfactory docking and molecular dynamics results compared to those obtained for Regadenoson (Lexiscan®), used as the positive control. These compounds can be used in biological assays (in vitro and in vivo) in order to confirm the potential activity agonist to A2AAR.

Adenosine Signaling Is Prognostic for Cancer Outcome and Has Predictive Utility for Immunotherapeutic Response.

PURPOSE: There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed. EXPERIMENTAL DESIGN: We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor-treated patients. RESULTS: The signature captures baseline adenosine levels in vivo (r 2 = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r 2 = -0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e-16) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e-16) and PFS (HR = 0.65, P = 0.0000002) in CD8+ T-cell-infiltrated tumors. Mutation of TGFß superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e-16). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012). CONCLUSIONS: These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.