Structural investigation on thiazolo[5,4-d]pyrimidines to obtain dual-acting blockers of CD73 and adenosine A2A receptor as potential antitumor agents

Adenosine pathway, including its generating enzyme (CD73) and its receptors represents a key target for cancer immunotherapy. Here we aimed to search for novel compounds able to co-target the CD73 and the A_2A adenosine receptor (A_2A AR) as dual-blockers of adenosine generation and activity. The design project was to combine in the same molecule the thiazolo[5,4-d]pyrimidine core, an essential pharmacophoric feature to block the A_2A AR, with a benzenesulfonamide group which is a characteristic group of CD73 inhibitors. Most of the reported compounds resulted in inverse agonists of the human (h) A_2A AR endowed with high affinity, selectivity and potency. However they were weak inhibitors of CD73 enzyme. Nevertheless, this study can be considered as a starting point to develop more active compounds.     

The PI3K–NF-κB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A₃ adenosine receptor (A₃AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A₃AR agonist, on adjuvant-induced arthritis.     

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport

A₁ adenosine receptors (ARs) reduce, and A₂ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A₁AR and A₂AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A₁, A_2A and A₃AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A₁ARs, CGS 21680 and DPMA for A_2AARs, and Cl-IB-MECA and IB-MECA for A₃ARs. Stimulation of A₁, A_2A and A₃ARs elevated Ca²⁺, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A₁, A_2A and A₃AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A₁, A_2A and A₃ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca²⁺ and volume, but likely by actions on separate cell targets. Reprint requests should be addressed to: Dr. Mortimer M. Civan, Dept. of Physiology, University of Pennsylvania, Richards Building, Philadelphia, PA 19104-6085. [Tel.: (215)-898-8773; Fax: (215)-573-5851]     

Adenosine,adenosine receptors and glaucoma: An updated overview

Background

Glaucoma, a leading cause of blindness worldwide, is an optic neuropathy commonly associated with elevated intraocular pressure (IOP). The major goals of glaucoma treatments are to lower IOP and protect retinal ganglion cells. It has been revealed recently that adenosine and adenosine receptors (ARs) have important roles in IOP modulation and neuroprotection.

Scope of review

This article reviews recent studies on the important roles of adenosine and ARs in aqueous humor formation and outflow facility, IOP and retinal neuroprotection.

Major conclusions

Adenosine and several adenosine derivatives increase and/or decrease IOP via A_2A AR. Activation of A₁ AR can reduce outflow resistance and thereby lower IOP, A₃ receptor antagonists prevent adenosine-induced activation of Cl⁻ channels of the ciliary non-pigmented epithelial cells and thereby lower IOP. A₁ and A_2A agonists can reduce vascular resistance and increase retina and optic nerve head blood flow. A₁ agonist and A_2A antagonist can enhance the recovery of retinal function after ischemia attack. Adenosine acting at A₃ receptors can attenuate the rise in calcium and retinal ganglion cells death accompanying P2X(7) receptor activation.

General significance

Evidence suggested that the adenosine system is one of the potential target systems for therapeutic approaches in glaucoma.     

CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth,angiogenesis, neovascularization,macrophage infiltration and metastasis

Ecto-5′-nucleotidase (CD73), an enzyme providing interstitial adenosine, mediates diverse physiological and pathological responses. In tumor progression, it has primarily an immunosuppressive role but is also thought to regulate neovascularization. However, the latter role is still in debate. When B16F10 melanoma was subcutaneously injected into CD73 knockout mice, changes in the tumor vasculature were not always observed. However, we demonstrated earlier that the growth and vascularization of B16F10 melanoma in CD73 knockout mice depend on the low presence of CD73 on tumor cells. To further analyze the role of CD73 on tumor growth and vascularization, we compared the changes in B16F10 melanoma subcutaneously injected into right flank of wild-type mice, CD73 knockout mice lacking host CD73 only, and CD73 knockout mice with tumor cell CD73 either inhibited with AOPCP (adenosine α,β-methylene 5′-diphosphate) or permanently knocked down through genetic modification. We report here that both inhibition and knockdown of tumor CD73 further inhibited tumor growth compared to host CD73 knockout alone. MAP-kinase signaling pathway activation also decreased more strongly in the stable knockdown. There was a significant reduction in the angiogenic activation of blood microvessels as observed by decreased anti-VEGFR2 staining. Stable CD73 knockdown also reduced endothelial cell proliferation as measured by anti-CD105 staining. However, only chemical inhibition with AOPCP significantly augmented the reduction in intratumoral microvessel density induced by host CD73 knockout. Such reduction was not observed when tumor CD73 was knocked down due to the much slower tumor growth and decreased oxygen demand as indicated by the low expression of Bad, a hypoxia marker. Decreased CD73 activity also led to the decreased expression of angiogenic factors, including VEGF and bFGF that was only partially reversed by hypoxia in tumors treated with AOPCP. Both inhibition and knockdown of tumor CD73 significantly decreased tumor macrophage infiltration and induced microenvironment changes, thereby influencing MI or MII macrophage polarization. Additionally, tumor cell CD73 is important in metastasis formation through adenosine-independent attachment to endothelium. We conclude that even low tumor cell CD73 expression has an undeniable role in melanoma progression, including the regulation of many aspects of angiogenesis. CD73 is thus a viable target in anti-angiogenic melanoma therapy.     

Effects of synthetic A3 adenosine receptor agonists on cell proliferation and viability are receptor independent at micromolar concentrations

The question as to whether A3 adenosine receptor (A3AR) agonists, N ⁶-(3-iodobenzyl)-adenosine-5′-N- methyluronamide (IB-MECA) and 2-chloro-N ⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), could exert cytotoxic effects at high concentrations with or without the involvement of A3AR has been a controversial issue for a long time. The initial findings suggesting that A3AR plays a crucial role in the induction of cell death upon treatment with micromolar concentrations of IB-MECA or Cl-IB-MECA were revised, however, the direct and unequivocal evidence is still missing. Therefore, the sensitivity of Chinese hamster ovary (CHO) cells transfected with human recombinant A3AR (A3-CHO) and their counter partner wild-type CHO cells, which do not express any of adenosine receptors, to micromolar concentrations of IB-MECA and Cl-IB-MECA was studied. We observed that IB-MECA and Cl-IB-MECA exhibited a strong inhibitory effect on cell proliferation due to the blockage of cell cycle progression at G1/S and G2/M transitions in both A3-CHO and CHO cells. Further analysis revealed that IB-MECA and Cl-IB-MECA attenuated the Erk1/2 signalling irrespectively to A3AR expression. In addition, Cl-IB-MECA induced massive cell death mainly with hallmarks of a necrosis in both cell lines. In contrast, IB-MECA affected cell viability only slightly independently of A3AR expression. IB-MECA induced cell death that exhibited apoptotic hallmarks. In general, the sensitivity of A3-CHO cells to micromolar concentrations of IB-MECA and Cl-IB-MECA was somewhat, but not significantly, higher than that observed in the CHO cells. These results strongly suggest that IB-MECA and Cl-IB-MECA exert cytotoxic effects at micromolar concentrations independently of A3AR expression.     

Differential inhibition of noradrenaline release mediated by inhibitory A1-adenosine receptors in the mesenteric vein and artery from normotensive and hypertensive rats

Mesenteric arteries and veins are densely innervated by sympathetic nerves and are crucial in the regulation of peripheral resistance and capacitance, respectively, thus, in the control of blood pressure. Presynaptic adenosine receptors are involved in vascular tonus regulation, by modulating noradrenaline release from vascular postganglionic sympathetic nerve endings. Some studies also suggest that adenosine receptors (AR) may have a role in hypertension. We aim at investigating the role of presynaptic adenosine receptors in mesenteric vessels and establish a relationship between their effects (in mesenteric vessels) and hypertension, using the spontaneously hypertensive rats (SHR) as a model of hypertension. Adenosine receptor-mediated modulation of noradrenaline release was investigated through the effects of selective agonists and antagonists on electrically-evoked [³H]-noradrenaline overflow. CPA (A₁AR selective agonist: 1–100 nM) inhibited tritium overflow, but the inhibition was lower in SHR mesenteric vessels. IB-MECA (A₃AR selective agonist: 1–100 nM) also inhibited tritium overflow but only in WKY mesenteric veins. CGS 21680 (A_2AAR selective agonist: up to 100 nM) failed to facilitate noradrenaline release in mesenteric veins, from both strains, but induced a similar facilitation in the mesenteric arteries. NECA (non-selective AR agonist: 1, 3 and 10 μM), in the presence of A₁ (DPCPX, 20 nM) and A₃ (MRS 1523, 1 μM) AR selective antagonists, failed to change tritium overflow. In summary, the modulatory effects mediated by presynaptic adenosine receptors were characterized, for the first time, in mesenteric vessels: a major inhibition exerted by the A₁ subtype in both vessels; a slight inhibition mediated by A₃ receptors in mesenteric vein; a facilitation mediated by A_2A receptors only in mesenteric artery (from both strains). The less efficient prejunctional adenosine receptor mediated inhibitory effects can contribute to an increase of noradrenaline in the synaptic cleft (both in arteries and veins), which might conduce to increased vascular reactivity.     

A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats

A_2A adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A_2AARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A_2AAR stimulation in a rat model of arthritis. We investigated A_2AAR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A_2AARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A_2AAR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A_2AAR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A_2AAR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A_2AAR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A_2AAR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A_2AARs in lymphocytes from RA patients. The effectiveness of A_2AAR stimulation in a rat model of arthritis supported the role of A_2AAR agonists as potential pharmacological treatment for RA.     

A3 adenosine receptor activation in melanoma cells: association between receptor fate and tumor growth inhibition

Activation of the Gi protein-coupled A3 adenosine receptor (A3AR) has been implicated in the inhibition of melanoma cell growth by deregulating protein kinase A and key components of the Wnt signaling pathway. Receptor activation results in internalization/recycling events that play an important role in turning on/off receptor-mediated signal transduction pathways. Thus, we hereby examined the association between receptor fate, receptor functionality, and tumor growth inhibition upon activation with the agonist 1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purine-9-yl]-N-methyl-beta-D-ribofuranuronamide (IB-MECA). Results showed that melanoma cells highly expressed A3AR on the cell surface, which was rapidly internalized to the cytosol and "sorted" to the endosomes for recycling and to the lysosomes for degradation. Receptor distribution in the lysosomes was consistent with the down-regulation of receptor protein expression and was followed by mRNA and protein resynthesis. At each stage, receptor functionality was evidenced by the modulation in cAMP level and the downstream effectors protein kinase A, glycogen synthase kinase-3beta, c-Myc, and cyclin D1. The A3AR antagonist MRS 1523 counteracted the internalization process as well as the modulation in the expression of the signaling proteins, demonstrating that the responses are A3AR-mediated. Supporting this notion are the in vivo studies showing tumor growth inhibition upon IB-MECA treatment and reverse of this response when IB-MECA was given in combination with MRS 1523. In addition, in melanoma tumor lesions derived from IB-MECA-treated mice, the expression level A3AR and the downstream key signaling proteins were modulated in the same pattern as was seen in vitro. Altogether, our observations tie the fate of A3AR to modulation of downstream molecular mechanisms leading to tumor growth inhibition both in vitro and in vivo.     

The pharmacological activation of adenosine A1 and A3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A₁ and A₃ receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N ⁶-cyclopentyladenosine (CPA), an adenosine A₁ receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N ⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A₃ receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.     

Deletion of CD73 increases exercise power in mice

Ecto-5′-nucleotidase or CD73 is the main source of extracellular adenosine involved in the activation of adenosine A_2A receptors, responsible for the ergogenic effects of caffeine. We now investigated the role of CD73 in exercise by comparing female wild-type (WT) and CD73 knockout (KO) mice in a treadmill-graded test to evaluate running power, oxygen uptake (V̇O₂), and respiratory exchange ratio (RER) — the gold standards characterizing physical performance. Spontaneous locomotion in the open field and submaximal running power and V̇O₂ in the treadmill were similar between CD73-KO and WT mice; V̇O₂max also demonstrated equivalent aerobic power, but CD73-KO mice displayed a 43.7 ± 4.2% larger critical power (large effect size, P < 0.05) and 3.8 ± 0.4% increase of maximum RER (small effect size, P < 0.05). Thus, KO of CD73 was ergogenic; i.e., it increased physical performance.     

Cardioprotective effects of adenosine A1 and A 3 receptor activation during hypoxia in isolated rat cardiac myocytes

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A₁ and A₃ receptors (A₁R and A₃R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4–5 days in vitro), to an atmosphere of a N₂ (95%) and CO₂ (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A₁ and A₃ receptor antagonists abolished the protective effects of adenosine. A₁R and A₃R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A₁R agonist, CCPA, was abolished by an A₁R antagonist, DPCPX, and was not affected by an A₃R antagonist, MRS1523. Cardioprotection caused by the A₃R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A₁R and A₃R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A₁ or A₃ adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A₁R and A₃R agonists may have potential as cardioprotective agents against ischemia or heart surgery.     

Adenosine protects against angiotensin II-induced apoptosis in rat cardiocyte cultures

Adenosine has been found to be cardioprotective during episodes of cardiac ischemia/reperfusion through activation of the A₁ and possibly A₃ receptors. Therefore, we have investigated whether activation of these receptors can protect also against apoptotic death induced by angiotensin II (Ang II) in neonatal rat cardiomyocyte cultures. Exposure to Ang II (10 nM) resulted in a 3-fold increase in programmed cell death (p < 0.05). Pretreatment with the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA, 1 M), abolished the effects of Ang II on programmed cardiomyocyte death. Moreover, exposure of cells to the A₁ adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) before pretreatment with CCPA, prevented the protective effect of the latter. Pretreatment with the A₃ adenosine receptor agonist N⁶-(3-iodobenzyl) adenosine-5-N-methyluronamide (IB-MECA, 0.1 M), led to a partial decrease in apoptotic rate induced by Ang II. Exposure of myocytes to Ang II caused an immediate increase in the concentration of intracellular free Ca²⁺ that lasted 40–60 sec. Pre-treatment of cells with CCPA or IB-MECA did not block Ang II-induced Ca²⁺ elevation. In conclusion, activation of adenosine A₁ receptors can protect the cardiac cells from apoptosis induced by Ang II, while activation of the adenosine A₃ receptors confers partial cardioprotection.     

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A_2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A₁ receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4–6 weeks old) and symptomatic (12–14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg²⁺ paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N⁶-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.     

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A_2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A₁, A_2A, and A_2B ARs. Cell proliferation was measured by [³H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A₁), BAY60-6583 (A_2B), and IB-MECA (A₃), and the antagonists PSB-36 (A₁), MSX-2 (A_2A), and PSB-10 (A₃) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A_2A), and the antagonists preladenant (SCH-420814, A_2A), PSB-1115 (A_2B), and PSB-603 (A_2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC₅₀ value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Ecto-5’-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model

Background

Ecto-5’-nucleotidase/CD73 (ecto-5’-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5’-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children.

Materials and Methods

The effects of ecto-5’-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified.

Results

The human MB cell line D283, transfected with ecto-5’-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A₁ adenosine receptor expression rates were detected in MB cells overexpressing ecto-5’-NT.

Conclusion

This work suggests that ecto-5’-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A₁ adenosine receptors. Therefore, ecto-5’-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.     

Adenosine A<Subscript>2A</Subscript> receptor and ecto-5′-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE)

Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A_2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A₁ receptors. Gain of function of the excitatory A_2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A_2A receptor and of the adenosine-generating enzyme, ecto-5′-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A_2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A_2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A_2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A_2A receptor and ecto-5′-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A_2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5′-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.