Rozprawy doktorskie na temat „Adenosine receptors”

Adenosine (Ado) is an endogenous nucleoside released from almost all cell types. It exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes A1, A2A, A2B and A3 (ARs). These receptors differ in their affinity for Ado, in the type of G protein that they recruit and finally in the downstream signalling that are activated in target cells. The levels of Ado in the interstitial fluid are in the range of 20-200 nM, but many pathophysiological conditions such as asthma, neurodegenerative disorders, chronic inflammatory diseases and cancer are associated with changes in Ado levels. The primary aim of Ado is to reduce tissue injury and promote repair by different receptor-mediated mechanisms, including the increase of oxygen supply/demand ratio, anti-inflammatory effects and stimulation of angiogenesis. The investigation of ARs and their ligands is a rapidly growing field; there is extensive evidence for the involvement of ARs in the physiological regulation of several homeostatic processes and their implication in the ethiology of many diseases. The aim of this work was to analyse the expression of ARs and the signalling pathway, transcription factors and cytochines activated by them in different pathophysiological conditions linked to hypoxic and inflammatory conditions. At first, the role of ARs was studied in healthy and aneuploid pregnancies to understand reasons of spontaneous abortion (SA). It has been suggested that the causes of SA in aneuploidy are no different to those in euploidy, with the increased frequency in the former perhaps being ascribable to a genetically-determined imbalance in the mediators of placental perfusion and uterine contraction. So aneuploidy can be used as a model of this event. The ado transduction cascade appears to be disturbed in Trisomy 21 (TR21) through reduced expression of A2BAR and A1AR. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A1AR and A2BAR could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing nitric oxide (NO) and vascular endothelial growth factor (VEGF). Next, the effects of ARs in glial cells under inflammatory conditions have been investigated. Glial cells, astrocytes and microglia, are important contributors to inflammatory immune responses and hypoxia-inducible factor 1 (HIF-1α) is the key transcription factor that is upregulated in response to hypoxia and inflammatory stimuli. Ado, through A1AR and A3AR activation, reduces lipopolysaccharide (LPS)-stimulated HIF-1α mRNA expression and protein accumulation by inhibiting LPS-triggered p42/p44 mitogen-activated protein kinase (p44/42 MAPK) and serine/threonine protein kinase (Akt) phosphorylation in normoxic and hypoxic conditions. This leads to an inhibition of genes involved in inflammation like Inducible Nitric Oxide Synthase (iNOS) and A2BARs, that are stimulated by LPS and further increased by LPS in concert with hypoxia, whilst does not affect angiogenesis and metabolic related genes, with the exception of glucose transporter 1 (GLUT1) and hexokinase 2 (HK2) that are stimulated by LPS in normoxia and reduced by A1AR and A3AR activation. These findings add a new molecular pathway activated by Ado in astrocytes to give a reduction of genes involved in inflammation and hypoxic injury that may cohexist in stroke, ischemia and other central nervous system (CNS) disorders.

Pyrazolopyrimidines are a general class of compounds which exhibit Aj adenosine receptor affmity. A number of pyrazolo(3,4-d)pyrimidine analogues of isoguanosine and i-methylisoguanosine has been synthesised. All compounds were tested forAi adenosine receptor affinity using a (311) R-PIA competitive binding assay. The N-i and N-5 positions were substituted with a number of different ailcyl and aryi groups. 3-Chiorophenyl substitution of the N-i position and butyl substitution of the N-5 position greatly enhanced the overall adenosine receptor affinity. Substitution by a methyl group at the N-7 position fixed the C-4 position in the imino tautomeric form. This resulted in a marked reduction in activity. The substitution of the N-2 position with a phenyl group produced an analogue with a similar structure to i,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX). A 2-phenyl substituent was favourable for interaction with the adenosine receptor. A number of pyrazolo(3,4-d)pyrirnidine analogues of 4,6-bis-a-carbamoylethylthio-i-phenylthiopyrazolo(3,4-d)pyrinhidine (DJB-KK) has also been synthesised and tested for Aj adenosine receptor affinity. 4,6-Bis-alkylthio-1-phenylpyrazolo(3,4-d)pyrimidines with a-carbamoylethyl and u-carbamoylpropyi groups were compared. The additional methyiene of the a-carbamoylpropyl group produced increased adenosine receptor affinity. 6-a-Carbamoylethylthio-4-mercapto-1-phenylpyrazolo(3,4-d)pyrimidine and 4-cc-carbamoylethylthio- i-phenylpyrazolo(3,4-dlpyrimidine were compared. Substitution of the C-6 position maintained activity, while substitution of the C-4 reduced activity.

Pyrazolopyrimidines are a general class of compounds which exhibit Aj adenosine receptor affmity. A number of pyrazolo(3,4-d)pyrimidine analogues of isoguanosine and i-methylisoguanosine has been synthesised. All compounds were tested forAi adenosine receptor affinity using a (311) R-PIA competitive binding assay. The N-i and N-5 positions were substituted with a number of different ailcyl and aryi groups. 3-Chiorophenyl substitution of the N-i position and butyl substitution of the N-5 position greatly enhanced the overall adenosine receptor affinity. Substitution by a methyl group at the N-7 position fixed the C-4 position in the imino tautomeric form. This resulted in a marked reduction in activity. The substitution of the N-2 position with a phenyl group produced an analogue with a similar structure to i,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX). A 2-phenyl substituent was favourable for interaction with the adenosine receptor. A number of pyrazolo(3,4-d)pyrirnidine analogues of 4,6-bis-a-carbamoylethylthio-i-phenylthiopyrazolo(3,4-d)pyrinhidine (DJB-KK) has also been synthesised and tested for Aj adenosine receptor affinity. 4,6-Bis-alkylthio-1-phenylpyrazolo(3,4-d)pyrimidines with a-carbamoylethyl and u-carbamoylpropyi groups were compared. The additional methyiene of the a-carbamoylpropyl group produced increased adenosine receptor affinity. 6-a-Carbamoylethylthio-4-mercapto-1-phenylpyrazolo(3,4-d)pyrimidine and 4-cc-carbamoylethylthio- i-phenylpyrazolo(3,4-dlpyrimidine were compared. Substitution of the C-6 position maintained activity, while substitution of the C-4 reduced activity.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Division of Science and Technology
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Na Anemia Falciforme em situações de baixa tensão de oxigênio, a hemoglobina mutante S (HbS) sofre polimerização promovendo a falcização das hemácias, que podem aderir ao endotélio vascular, causando a oclusão de vasos (VO) e isquemia tecidual (crises dolorosas) que caracterizam o quadro clínico da doença. Além disso, os pacientes falciformes apresentam outras manifestações clínicas como o priapismo, alterações ósseas, certas complicações pulmonares entre outros. Além das células eritróides, células endoteliais, leucócitos e plaquetas também desempenham um papel fundamental na fisiopatologia da anemia falciforme. A hidroxiuréia (HU), na anemia falciforme, aumenta a produção de hemoglobina fetal (HbF) em células eritróides, reduzindo a polimerização da HbS, diminuindo os sintomas clínicos dos pacientes. O aumento da HbF, no entanto, não implica necessariamente na melhora clínica, indicando desta forma a potencial ação da HU sobre outros processos. Estudos recentes vêm relacionando priapismo e asma com elevados níveis de adenosina. Devido a esta importância da adenosina relacionada a patologias comuns a AF, tivemos como objetivo identificar polimorfismos em genes de receptores de adenosina e na adenosina deaminase e verificar a possível associação entre as manifestações clínicas, além de investigar o papel da HU na modulação de marcadores envolvido na síntese e degradação da adenosina. Foram analisados diversos sítios polimórficos nos genes que codificam ADORA1, ADORA 2b, ADORA 3 e ADA, seguindo com a genotipagem em pacientes com AF, comparando afetados e não afetados. Em adição foi avaliada a expressão diferencial de mRNA de ADA pela HU em monócitos destes pacientes, comparando tratados e não tratados e também avaliamos por citometria de fluxo a modulação de marcadores de superfície CD39, CD73 e CD26, pela HU. As análises estatísticas foram realizadas utilizando os softwares GenePop 3.4 para análises de associação, cálculo do HWE, GraphPad Prism 5, Arlequin para identificação de desequilíbrio de ligação, haplótipos, heterozigozidade e SAS 9.13 para associação dos haplótipos as características. Os resultados mostraram que os pacientes sob tratamento com HU apresentaram um aumento da expressão de mRNA de ADA, aumento da expressão de CD26 em monócitos e diminuição de CD39 em linfócitos. Sem alterações significativas em relação a CD73. Encontramos também um aumento da freqüência do alelo T do SNP (rs1685103) presente no gene de ADORA 1 associado com pacientes afetados com síndrome torácica aguda. Apesar de não ter sido estatisticamente significante, concorda com dados da literatura. No gene ADORA 2B, verificamos associação do SNP 1007 C>T no desenvolvimento de STA indicando o alelo T como fator de risco e o alelo C para alterações ósseas. Para o SNP 968 G>T houve associação com alterações ósseas. Na análise haplotípica entre os SNPs 968 G>T e 1007 C>T encontramos associação dos haplótipos ht2 e ht3 com STA, como fator de risco, ht2 para hipertensão pulmonar. ht1 para priapismo, alterações ósseas e estenose/AVC. Os haplótipos formados pelos três SNPs 968 G>T, 1007 C>T e rs16851030, encontramos associação entre ht1, ht3 e ht4 entre os afetados com priapismo, caracterizando-o como haplótipo de risco e também ht1 e ht6 associados à estenose/AVC. Concluímos, que a hidroxiuréia participa na modulação da expressão da adenosina deaminase, de CD26 em monócitos e CD39 em linfócitos. Além disso, mostrou-se a importância de sítios polimórfico presente no gene ADORA 2B e ADORA1 envolvido na fisiopatologia das manifestações clínicas da doença falciforme. Associações dos SNPs em ADORA 1 e ADA, devem ser melhor estudados em um número maior de pacientes. A determinação destes polimorfismos associados com diferentes características clínicas pode levar a um melhor entendimento dos processos fisiopatológicos da anemia falciforme, levando à identificação de pacientes de risco, possibilitando um manejamento racional dos mesmos, em termos de cuidados específicos, ou mesmo à determinação de alvos para o desenvolvimento de terapias alternativas.
In sickle cell disease in low oxygen tension, mutant hemoglobin S (HbS) undergoes polymerization promoting sickling of red blood cells that can adhere to vascular endothelium, causing vessel occlusion (VO) and tissue ischemia (painful crises) that characterize the clinical disease. In addition, sickle cell patients have other clinical manifestations such as priapism, bone disorders, certain pulmonary complications among others. In addition to the erythroid cells, endothelial cells, white cells and platelets also play a key role in the pathophysiology of sickle cell anemia. Hydroxyurea (HU) in sickle cell anemia, increases the production of fetal hemoglobin (HbF) in erythroid cells, reducing the HbS polymerization, reducing the clinical symptoms of patients. The increase in HbF, however, does not necessarily imply clinical improvement, thus indicating the potential effects of HU on other processes. Recent studies relating asthma and priapism with high levels of adenosine. Due to this importance of adenosine-related pathologies common to AF, we aimed to identify gene polymorphisms in adenosine receptors and adenosine deaminase and verify the possible association between clinical manifestations, and to investigate the role of HU in the modulation of markers involved synthesis and degradation of adenosine. We analyzed several polymorphic sites in genes that encode ADORA1, ADORA 2b, 3 and ADORA ADA, according to the genotype in patients with AF, comparing affected and unaffected. In addition we assessed the differential expression of ADA mRNA by HU in monocytes of these patients, comparing treated and untreated, and also evaluated by flow cytometry modulation of surface markers CD39, CD73 and CD26 by HU. Statistical analysis was performed using the software GenePop 3.4 for association analysis, calculation of HWE, GraphPad Prism 5, Arlequin for identification of linkage disequilibrium, haplotypes, heterozygosity and SAS 9.13 for association of haplotypes features. The results showed that patients treated with HU showed an increase in mRNA expression of ADA, increased expression of CD26 on monocytes and decreased CD39 on lymphocytes. No significant changes in relation to CD73. We also found an increased frequency of allele T (SNP rs1685103) present in a gene associated with ADORA affected patients with acute chest syndrome. Although not statistically significant, agrees with literature data. ADORA 2B gene, we found association of the SNP 1007 C> T in the development of STA indicating the T allele as a risk factor for the C allele and bone changes. For the SNP 968 G> T was associated with bone disorders. In haplotype analysis between SNPs 968 G> T and 1007 C> T found association of haplotypes ht2 and HT3 with STA as a risk factor for pulmonary hypertension ht2. ht1 for priapism, stenosis and bone disorders / stroke. The three haplotypes formed by SNPs 968 G> T, 1007 C> T and rs16851030, we found association between ht1, HT3 and HT4 among those affected with priapism, characterizing it as a risk haplotype and also ht1 ht6 associated with renal and / AVC. We conclude that hydroxyurea participates in modulating the expression of adenosine deaminase of CD26 on monocytes and CD39 on lymphocytes. Moreover, he showed the importance of polymorphic sites in this gene and ADORA 2B ADORA1 involved in the pathophysiology of clinical manifestations of sickle cell disease. Associations of SNPs in ADORA 1 and ADA should be better studied in a larger number of patients. The determination of these polymorphisms associated with different clinical characteristics can lead to a better understanding of the pathophysiological processes of sickle cell anemia, leading to the identification of patients at risk, enabling a rational handling of the same in terms of specific care, or even the determination of targets for the development of alternative therapies.

Impaired wound healing is a common and serious complication of diabetes mellitus causing an increased risk of infection and tissue damage. Chronic wounds associated with diabetes may be caused by peripheral neuropathy, vascular complications and alterations in immune function and contributes to non-traumatic amputations. Consequently, therapeutic management of wounds in diabetes is centred on infection control and stimulating revascularization. This research will focus on the latter and investigate the effects of high glucose on endothelial function with a view of finding new strategies to improve wound healing in diabetes. This project identified the adenosine receptor subtypes in EA.hy926 endothelial cell lines that are involved in stimulating wound healing and determined whether their roles are affected in the hyperglycaemic environment associated with diabetes. As caffeine is a non-selective antagonist of the adenosine receptors, this study also investigated the effects of acute and 48 hour chronic caffeine treatments on adenosine receptor populations and on wound closure and cell proliferation. Caffeine has a crucial role in wound healing through various mechanisms such as antioxidant activity or increasing the level of cyclic adenosine monophosphate (cAMP).
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor.

This thesis describes investigations into the tissue distribution of messenger RNAs encoding the four cloned adenosine receptors in the rat, and aspects of adenosine A₁ and A_2a receptor function in the rat central nervous system (CNS). Chapter 1 reviews the literature relating to this field, presenting an overview of adenosine metabolism and adenosine receptor classification, distribution and function. In Chapter 2 the distribution of adenosine A₂ and A₃ receptor gene expression is described. Chapter 3 describes investigations into the possible anatomical association between the adenosine A₁ receptor and members of Kir3.0, a subfamily of inwardly rectifying K⁺ channels. In Chapter 4 adenosine A₁ and A₂ receptor mediated control of proenkephalin and substance P gene expression in the rat striatum was investigated. In Chapter 5 the results of an investigation into interactions between striatal adenosine A₁ and A_2a, receptors, is described. Chapter 6 describes work performed on a related project in which a technique was developed to promote the fusion of cortical synaptosomes, thus rendering them amenable to elctrophysiological examination. The fused entities resulting from this procedure were subsequently characterised. In conclusion, the tissue distribution, at the molecular level, of all four cloned adenosine receptors has been defined. Consequently many novel sites of adenosine receptor gene expression have been identified. Also, certain functional effects of the striatal adenosine A_2a receptor have been demonstrated for the first time, these include an ability to control the activity of both major striatal output pathways and a modulatory interaction with colocalised adenosine A₁ receptors. The latter observations may be of some importance in the development of new therapeutic approaches designed to alleviate the symptoms of Parkinson's disease.

Adenosine receptors that mediate vasodilatation on the rat basilar artery are determined, and the transduction mechanisms by which they mediate their relaxations investigated. Furthermore, the effect of reactive oxygen species and hypoxia upon this vessel are described. Dilatation of the rat basilar artery by adenosine receptors is a complex process involving more than one receptor. The functional data obtained using the small vessel myograph are consistent with the combined action of at least three adenosine receptors. The adenosine receptor agonist, NECA, acts upon A₂ receptors in the lower concentration range (< 10 μM) of its biphasic curve whilst the higher concentration range is xanthine-insensitive, suggesting the action of the A₃ receptor. R-PIA also acts upon the xanthine-insensitive receptor whilst IB-MECA and APNEA would appear to be acting via A₂ receptors. Using reverse transcription with the polymerase chain reaction, all four adenosine receptors were shown to be expressed in groups of rat dissociated basilar artery myocytes. The A₁ receptor is expressed but does not mediate vasodilatation. The first phase of the NECA curve mediates relaxation by positively coupling to adenylyl cyclase, shown by the blockade of relaxations in the lower concentrations range of the NECA curve by the protein kinase inhibitor Rp-cAMPS. R-PIA would appear to act upon the A₃ receptor mediating relaxation, in part, via NO release as shown by shifting of the concentration-effect curve to R-PIA in the absence of endothelium and the presence of L-NAME. The second mechanism by which R-PIA mediates its relaxation was not determined but is not via potassium channels, adenylyl cyclase, release of 5-HT and histamine from mast cells nor prostanoid release. Investigation into the mRNA encoding for adenylyl cyclase isotypes revealed the presence of multiple isoforms on the rat basilar artery, namely Types II, III, IV, V, VI, VIII with a splice variant detected for Type IV.

G protein-coupled receptors (GPCRs) elicit an ability to activate multiple downstream signalling pathways. It is becoming evident that for many GPCRs, agonists are able to activate several of these pathways, each to differing extents; a phenomenon termed pathway bias, or biased agonism. Here, work is presented quantifying biased agonism for the: adenosine A1 and A2A receptors, as well as the calcitonin-like receptor (CLR). For the adenosine receptors, novel selective, and non-selective, agonists are identified and characterised. Further, the extent of biased agonism is determined for A1R agonists with respect to their abilities to positively and negatively regulate cAMP production, mobilise intracellular Ca2+ and activate ERK1/2. The activity of triazoloquinazoline compounds against the A2AR is validated, identifying 3 to be selective. Further investigations into the ability of triazoloquinazolines to mediate cAMP production and ERK1/2 activation uncovers each tested agonist to be biased towards activating ERK1/2, at the A2AR. A characterisation of the effects of receptor activity modifying proteins (RAMPs) upon signalling from the CLR is presented: quantifying the extent of biased agonism, with respect to the ability of RAMP-CLR heterodimers to: mediate cAMP production and inhibition, as well as mobilise intracellular Ca2+, uncovering this to be a Gαq/11-mediated process. Further, through applying a saturation mutagenesis approach to the CLR, a potential interaction is identified between intracellular loop 1 (ICL1) and helix 8, which is broken upon receptor activation, further identifying ICL1 to be a region of the CLR responsible for influencing G protein specificity. Ultimately, these findings relating to both adenosine and CLR-based receptors uncovers further evidence of biased agonism at GPCRs, which may have potential implications upon improving the efficacy and safety profiles of novel pharmaceutics targeting these clinically relevant GPCRs.

The neuromodulator adenosine affects a wide variety of physiopathological processes through activation of four receptors, classified as A1, A2A, A2B, and A3 subtypes. Adenosine receptors (ARs) belong to family A of G protein-coupled receptors (GPCRs) and are ubiquitously expressed in the human body. Activation or blockade of ARs is responsible for a wide range of effects in numerous organ systems; and therefore the regulation of ARs can have many potential therapeutic applications. The main objective of this project has been the investigation of the in silico molecular pharmacology of adenosine receptors and, in particular, of the human A2A and A3 adenosine receptors to guide the discovery and the structural refinement of new potent and selective AR antagonists. The recently published crystal structures of the human A2A adenosine receptor (hA2AAR) provide detailed three-dimensional information useful to support homology modeling studies and receptor-based drug design approaches. In particular, the 2.6 Å crystallographic structure of the hA2AAR in complex with the potent and selective antagonist ZM241385 was used as template to build a homology model of the hA3AR. In order to validate the molecular docking protocols for the adenosine receptors family, the hA2AAR crystal structure was used to perform in parallel molecular docking studies using different docking software. Then RMSD values between predicted and crystallographic poses of ZM241385 were calculated to select the docking protocol able to better reproduce this molecular system and to be used in the following molecular docking studies. Subsequently, molecular docking studies of different ARs antagonists were performed at the hA3AR model and at the hA2AAR crystal structure, enabling the exploration of the potential effects of chemical modifications of these compounds, and thus facilitating the lead optimization process. Different series of new compounds belonging to known adenosine antagonists classes, including triazolo-triazines and pyrazolo-triazolo-pyrimidines, have been analyzed and modified with the aim to modulate their affinity towards different adenosine receptor subtypes, to increase their solubility, or to overcome their metabolic instability. Moreover, several compounds with simplified scaffolds have been proposed as new adenosine receptor antagonists; such as pyrazolo-pyrimidinones, phthalazinones and triazolo-pyrimidines. Finally, the knowledge gained through the docking studies led to the identification of structural features of antagonist compounds important for the interaction with the hA3AR and was applied to the design of fluorescent ligands for this subtype, of particular interest as pharmacological probes. In conclusion, the integration of in silico studies with synthetic work and pharmacological tests resulted to be a good strategy for the development of new compounds as adenosine receptors antagonists and led to a better understanding at the molecular level of this class of GPCRs.
L’adenosina è un neuromodulatore che regola molti processi fisiopatologici attraverso l’attivazione di quattro diversi recettori accoppiati a proteine G (GPCRs), classificati come sottotipi A1, A2A, A2B e A3. I recettori adenosinici sono ubiquitari nell’organismo umano e la loro attivazione è responsabile di numerosi effetti in diversi organi. Proprio per questo motivo la regolazione dell’attività di questi recettori può avere interessanti applicazioni terapeutiche. Il principale obiettivo di questo progetto è stato l’analisi in silico a livello molecolare dei recettori adenosinici, ed in particolare dei recettori adenosinici umani A2A e A3, per guidare la scoperta e l’ottimizzazione strutturale di nuovi antagonisti adenosinici potenti e selettivi. Le strutture cristallografiche del recettore adenosinico umano A2A, recentemente pubblicate, forniscono dettagliate informazioni strutturali utili per supportare studi di homology modeling e approcci di drug design di tipo structure-based. In particolare, la struttura cristallografica del recettore adenosinico umano A2A, in complesso con l’antagonista potente e selettivo ZM241385, è stata utilizzata come templato per la costruzione di un modello per omologia del recettore adenosinico umano A3. Inoltre, con l’intento di selezionare il protocollo di docking molecolare più adatto per la famiglia dei recettori adenosinici, la struttura cristallografica del recettore adenosinico A2A è stata utilizzata per effettuare simulazioni di docking con diversi softwares in parallelo. Successivamente, le conformazioni ottenute dal docking sono state confrontate con la pose cristallografica di ZM241385 per selezionare il protocollo di docking che fosse in grado di riprodurre al meglio questo sistema molecolare e che potesse quindi essere usato per i successivi studi di docking. Sono stati quindi effettuati studi di docking molecolare di vari antagonisti adenosinici sul modello del recettore A3 e sulla struttura cristallografica del recettore A2A, in modo da ricavare informazioni che potessero facilitare il processo di ottimizzazione dei composti. Sono stati infatti analizzati numerosi nuovi composti appartenenti a classi note di antagonisti adenosinici, tra cui composti triazolotriazinici e tirazolotriazolopirimidinici, in modo da suggerire modifiche strutturali in grado di modularne l’affinità nei confronti dei vari sottotipi recettoriali adenosinici, di aumentarne la solubilità o di superarne i punti di instabilità metabolica. Diversi derivati con strutture semplificate, come per esempio composti pirazolopirimidinonici, ftalazinonici e triazolotriazinici, sono stati inoltre proposti come nuovi composti con attività antagonista nei confronti dei recettori adenosinici. Le informazioni ricavate grazie agli studi di docking hanno permesso l’identificazione di caratteristiche strutturali degli antagonisti adenosinici fondamentali per l’interazione con questi recettori. Queste informazioni sono state quindi applicate alla progettazione di derivati fluorescenti per il recettore adenosinico A3, che risultano particolarmente interessanti per il loro potenziale utilizzo in saggi farmacologici. In conclusione, quindi, questo studio sui recettori adenosinici dimostra come l’integrazione di metodologie computazionali con il lavoro sintetico e farmacologico risulta essere una strategia efficace per lo sviluppo di nuovi ligandi dei recettori adenosinici, a potenziale interesse terapeutico, e per il chiarimento di importanti aspetti strutturali riguardanti questa famiglia recettoriale e più in generale tutti i GPCRs.

Abstract Adenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A3 receptor (A3R) is the least explored among the four human adenosine receptor subtype members (A1, A2A, A2B and A3) and it is implicated in both neuroprotective and neurodegenerative effects. During the course of this work, the production of the recombinant human A3R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D2 receptor (D2R) was studied. A3R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A3R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A3R expression: 10µg of the monomeric A3R could be purified from one liter of insect cell culture. For successful crystallization thermostability of the A3R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A3R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D2R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D2R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D2R showed that the protein-protein interactions are calcium dependent
Tiivistelmä Adenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A3 reseptori (A3R) on adenosiinireseptorien neljästä alatyypistä (A1, A2A, A2B ja A3) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin. Tässä työssä arvioitiin sekä ihmisen rekombinantti-A3R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D2 reseptorin (D2R) heteromerisoitumista. Rekombinantti A3 reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA3R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A3R:n tuottoon: noin 10 µg monomeerista A3R:a voitiin puhdistaa litran hyönteissoluviljelmästä. Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A3R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A3R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A3R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille. Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D2R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D2R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D2R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen

The adenosinergic system is a neuromodulatory complex acting on four receptor subtypes with varying distributions throughout the central nervous system. This system contributes to homeostasis and regulates several physiological functions including sleep, motor function, synaptic plasticity or cognition. Adenosine can exert an upstream modulation of several neurotransmitter systems, including dopamine and glutamate, both implicated in the pathophysiology of neuropsychiatric diseases and particularly in psychosis. Schizophrenia is considered a psychotic disease characterized by a chronic and debilitating course that can be triggered by different genetic, developmental and environmental factors. The disease is manifested with three symptom domains including positive, negative and cognitive symptoms and has important treatment limitations. Preclinical and clinical data suggest that adenosine dysfunction may contribute to the pathophysiology of schizophrenia. Hence, the main focus of this thesis is to study the responses induced by the complete deletion of adenosine A2A receptors in the pathophysiology of the disease, paying special attention to the cognitive symptom domain. The use of biochemical and behavioural approaches has allowed us to assess the validity of A2A receptor KO as a possible animal model for the study of schizophrenia.
El sistema adenosinèrgic és un neuromodulador que actua sobre quatre subtipus de receptors amb diferents distribucions en el sistema nerviós central. Aquest sistema contribueix a l’homeòstasi i a la vegada regula diverses funcions fisiològiques com per exemple la son, la funció motora, la plasticitat sinàptica o la cognició. L’adenosina pot exercir un control “upstream” sobre diferents sistemes de neurotransmissió, incloent la dopamina i el glutamat, neurotransmissors implicats en la fisiopatologia de les malalties neuropsiquiàtriques, especialment en la psicosis. L’esquizofrènia es considera una malaltia psicòtica que cursa de forma crònica i debilitant provocada per la interacció de diferents factors genètics, ambientals i del desenvolupament. La malaltia es manifesta en tres dominis de símptomes, incloent símptomes positius, negatius i cognitius, i actualment té importants limitacions terapèutiques. Estudis preclínics i clínics suggereixen que la disfunció en el sistema d’adenosina pot contribuir a la patofisiologia de l’esquizofrènia. Per tant, l’objectiu principal d’aquesta tesi és l’estudi dels efectes induïts per la supressió completa dels receptors A2A d’adenosina en la fisiopatologia de la malaltia, parant especial atenció al domini de símptomes cognitius. L’ús de mètodes bioquímics i comportamentals ens ha permès avaluar la validesa dels KO pel receptor A2A d’adenosina com a possible model animal per a l’estudi de l’esquizofrènia.

Catecholamines, including dopamine (DA) and norepinephrine (NE), are widely distributed in the body and constitute a class of conventional neurotransmitters and hormones that occupy key positions in the regulation of physiological processes and in the development of neurological, psychiatric, endocrine and cardiovascular diseases. There is a linkage between a variety of genes related to DA (e.g. D4 receptor) and NE (e.g. α2A-adrenoceptor) and the vulnerability for developing attention deficit hyperactivity disorder (ADHD), which is characterized by pervasive symptoms of inattention, impulsivity and/or hyperactivity. In addition, adenosine, acting on adenosine receptors (AR), is a modulator of other receptors such as D1-like and D2-like DA receptors (DR). DA and adenosine receptor complexes are involved in the control of the direct and indirect pathway of motor control in basal ganglia, in which adrenergic receptors are also involved. NE, DA and adenosine receptors belong to the GPCR family, also known as seven transmembrane domain receptors. GPCRs have an enormous biomedical importance. It is estimated that about 35% of approved drugs target GPCRs. Thus, is not surprising that lots of models have been developed in order to explain the pharmacological behavior of these receptors. A large number of GPCRs have been described to form homodimers, heterodimers and higher order oligomers with different pharmacological and functional properties than of its individual components. The aim of this Thesis has been to study and characterize the molecular interactions at pharmacological and functional level of heterodimers between catecholamine receptors and between catecholamine and adenosine receptors involved in several neurological pathologies related to imbalances in attention, impulsivity and motor control. For simplicity, for pharmacologically characterize GPCRs, most of the developed models consider them as monomeric entities. Thus, it is not surprising that, when working with receptor dimers but using monomeric models, some parameters obtained may be erroneous. Then, first of all, we went deeper into the study of allosteric interactions within GPCRs using the dimer receptor model, which considers GPCRs as dimers. Along our study, we have given further evidences that homodimers are the GPCR predominant species, and that allosteric interactions between orthosteric ligands of the different protomers of a GPCR heteromer, have important implications in the field of catecholamine receptor pharmacology. A paradigmatic example of complex allosteric interactions is the A2AR-A2AR-D2R-D2R-Gs-Gi-AC5 heteromer. Moreover, since bivalent ligands are the best example of oligomer selective-ligands that can interact simultaneously with GPCR dimers with high affinity and subtype selectivity, we have developed a precise strategy for developing them. Using computational tools that considered the TM interfaces, distances between orthosteric binding sites and the mode of interaction of the pharmacophore units, we have higher success in affinity results. In particular, the obtained GPCR bivalent ligand had a picomolar binding affinity for the dopamine D2 receptor (D2R) homodimer. However, to obtain oligomer-selective bivalent ligands, the selected pharmacophores must be highly specific. This is not always easy to find. As an example, we have demonstrated that catecholamine receptors constitute a “functional” family of GPCRs. Specifically, in this study we have shown that DA and synthetic DA receptor ligands are able to bind to α2Rs and activate the same signaling pathways as NE. In addition, we have demonstrated the existence of functional D4R-D2SR in vitro and, for the first time, functional D4R-α2AR heteromers in vitro and in rodent brain tissues not only with the D4.4R but also with the D4.7R variant, prevalent in ADHD. Significant different properties of these heteromers were D4R variant-dependent. Finally, given that D2R, D4R and α2AR, are involved in the pathophysiology of ADHD, we suggest that D4R-D2R and α2AR-D4R heteromers could be target for the therapeutic treatment of such neurological disorders.
Les catecolamines dopamina (DA) i norepinefrina (NE) tenen una funció clau en la regulació de processos fisiològics i en el desenvolupament de diverses patologies. Existeix una correlació entre gens relacionats amb la DA (com el receptor D4) i amb la NE (com el receptor α2A) i la vulnerabilitat per desenvolupar el trastorn per dèficit d’atenció i hiperactivitat (TDAH). A més, l’adenosina, actuant sobre els receptors d’adenosina (AR), és un modulador dels receptors de DA tipus D1 i D2, que controlen el moviment als ganglis basals, on també es troben implicats els adrenoreceptors. Els receptors de NE, DA i adenosina pertanyen a la família dels GPCR, que té una gran importància biomèdica, essent diana d’un 35% dels fàrmacs aprovats. És conegut que els GPCRs formen homodimers, heterodimers i oligòmers més complexos amb noves propietats farmacològiques i funcionals. En aquesta Tesi hem caracteritzat les interaccions moleculars entre receptors de catecolamines i entre receptors de catecolamines i d’adenosina, involucrats en patologies neurològiques relacionades amb l’atenció, la impulsivitat i el control motor. Concretament, hem donat evidències que els homodimers de GPCRs són les espècies predominants a l’organisme i que les interaccions alostèriques entre lligands ortostèrics dins un heteròmer tenen importants implicacions farmacològiques. També hem generat un protocol de síntesis de lligands bivalents molt eficient. Aquests lligands permeten actuar sobre un oligòmer concret, minimitzant els efectes secundaris en comparació amb fàrmacs dirigits a monomèrs. Hem demostrat que els receptors de catecolamines constitueixen una mateixa família funcional donada la promiscuïtat entre els seus lligands. Finalment, hem descrit l’existència de complexos entre els receptors D4 i D2S i entre D4 i α2A, trobant diferències funcionals segons la variant del receptor D4 involucrada. Donat que els receptors D2R, D4R i α2AR estan involucrats en el TDAH, aquests heteròmers poden ser una nova diana terapèutica per a aquesta patologia.

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Adenosine is a purine nucleoside that is commonly expressed in the body and involved in a wide range of physiological processes. The effects of adenosine are mediated through specific G-protein coupled cell surface receptors, named A1, A2A, A2B and A3 adenosine receptors (ARs). It is now well recognized that extracellular levels of adenosine markedly increase under metabolically stressful conditions and in the last two decades it has become clear that adenosine is a mediator involved in the pathogenesis of lung inflammatory disorders and in cancer. Indeed adenosine induces bronchoconstriction in animal models and in patients with airway diseases such as asthma and chronic obstructive pulmonary diseases (COPD). COPD is defined as an inflammatory respiratory disease, largely caused by exposure to tobacco smoke. The disease is characterized by a progressive and incompletely reversible airflow obstruction. The key elements of COPD are exposure to cigarette smoke, airway inflammation, and airflow obstruction that is not fully reversible. In addition, an high concentration of adenosine have been reported in cancer tissues, where it appears to be implicated in the growth of tumors, and in the regulation of cell death and proliferation. The development of pleural-based abnormalities such as malignant pleural mesothelioma (MPM), is caused by inhalation of asbestos fibers. To study the interaction between adenosine and pulmonary diseases, the presence and functionality of ARs were explored in human healthy subjects in comparison with COPD patients by using lung tissues and bronchoalveolar lavage (BAL). The aim of this study was to investigate the expression, affinity and density of ARs in peripheral lung parenchyma and in BAL from age-matched smokers with COPD and smokers with normal lung function. The data, obtained in lung samples from COPD patients, suggest a downregulation of A2BARs and an upregulation of A2A and A3ARs. A correlation among ARs expression and clinical parameters such as FEV1/FVC ratio, an established index of airflow obstruction, was observed. Moreover the subcellular location of A2BARs in BAL macrophages of patients with COPD and healthy smokers was investigated, revealing a significant decrease in A2BARs expression in BAL from COPD patients compared to an age-matched group of control smokers with normal lung function. This reduction was associated with a decrease in A2BARs mRNA expression, suggesting a regulation at the transcriptional level. ARs were also studied in MPM patients by using membranes or primary cultures from pleura of MPM patients in comparison with healthy tissues or cells. Furthermore, to better understand the interaction between adenosine and MPM, the presence and the functionality of ARs were explored in healthy mesothelial cells (HMC) and malignant mesothelioma cells (MMC). ARs were analyzed by using RT-PCR, western blotting and saturation binding assays. HMC were treated with crocidolite asbestos, which is the principal risk factor of MPM. The dual effect of ARs stimulation on healthy and cancer cell growth was studied by means of proliferation, apoptosis and cytotoxicity assays. The main result was that A3ARs were up-regulated in MPM when compared with healthy mesothelial pleura. Stimulation of A3ARs decreases proliferation and exerts cytotoxic and pro-apoptotic effect on MMC and on HMC exposed to asbestos and TNF-�, but not in control HMC. These data support the potential for modulating ARs function in pulmonary diseases such as inflammation and cancer. In particular, the modulation of A2BARs by using selective antagonists in alveolar macrophages as a new potential pharmacological tool could be used in the COPD disease. Moreover, the stimulation of A3ARs by selective agonists could be involved as novel pharmacological treatment for malignant pleural mesothelioma.

Adenosine (Ado) is involved in the regulation of many physiological and pathophysiological processes through the activation of four cell membrane receptors termed A1, A2A, A2B and A3, which belong to the family of G-proteins coupled receptors and are ubiquitously expressed in the body. Adenosine receptors (ARs) are present on virtually every tissue and are often co-expressed in the same cell type, and their activation is predominantly responsible for the wide variety of effects produced by adenosine throughout several organ systems. Research on adenosine receptors during the past 20 years has resulted in considerable progress in identifying selective agonists and antagonists and an increased understanding of the particular roles adenosine receptor subtypes play in physiological processes. With this research work new ligands for the ARs have been synthesized and tested on A1, A2A, A2B and A3 subtypes. Hence, in the search for agonists for the A2B AR subtypes, 2-phenylhydroxypropynyl-5'-N-alkylcarboxamidoadenosine derivatives and N6-substituted 2-phenylhyroxypropynyladenosine derivatives were synthesized on the basis that the introduction of a phenylhydroxypropynyl chains in 2-position of Ado derivatives resulted in compounds with reasonably good A2B potency. Adenylyl cyclase assay, performed with cloned human A2B ARs, showed that these modifications did not produce the expected increase of potency at A2B receptors. Furthermore, in the search for A3 AR agonists, the synthesis of 2-phenylethynylAdo derivatives, bearing different substituents in the phenyl ring of the alkynyl chain and a methoxy group in the N6-position, was undertaken on the bases that these modifications are reported to favour the interaction of Ado derivatives with the A3 AR subtype. Selective compounds were also modified in the 4' position of the sugar moiety by introduction of a methyl or an ethyl carboxamido substituent. The new 2-aralkynyl-N6-methoxyAdo derivatives were found to possess high affinity and in some cases very high selectivity for the human A3 receptor subtype. Finally, based from the observation that the replacement of the sugar moiety in adenosine derivatives with alkyl groups led to adenosine receptor antagonists, in the last part of this work, the ribose moiety of Ado has been substituted with a ethyl group and alkynyl chains were introduced in the 2- and 8- positions, respectively. Binding studies (A1, A2A, A3) and adenylyl cyclase assay (A2B) performed at both human and rat ARs showed that all the synthesized compounds bound the ARs with good affinity. It is worthwhile to note that, in the series of the 2-substituted derivatives, the 2-cyclohexylethynyl-9-ethyladenine and 2-phenylhydrxypropynyl-9-ethyladenine are the ones endowed with the highest affinity at both human and rat A1 and A2A receptors. The same trend has been observed with the 8-substituted derivatives: in fact the 8-phenylethynyl-9-ethyladenine resulted the most active and selective compound at both human and rat A3 subtype. From these results it is possible to conclude that the selectivity of these two series of derivatives for the A1, A2A, and A3 receptor subtypes is strictly correlated to the nature of the alkynyl chain, and that there is a good correlation between the affinity at human and rat receptors.

Adenosine is an endogenous purine nucleoside that is constitutively present at low levels outside the cells but might dramatically increase its concentrations following metabolic stress conditions like those induced by hypoxia or ischaemia. After its release adenosine induces its biological effects through the interaction with four cell surface receptors classified by molecular, biochemical and pharmacological data into four subtypes: A1, A2A, A2B and A3. Adenosine through the interaction with A2 and A3 receptors plays a crucial role in inflammation and in the regulation of immune cells. A2A receptors, proposed as “natural” brakes of inflammation, appear to represent a promising pharmacological target to treat a wide variety of diseases characterized by a strong immunoinflammatory component. On the other hand, it may be advantageous in some circumstances to enhance certain aspects of inflammation in order to eliminate the causative agent, as in the case of cancer. In fact, it has to be remarked that tumour defence mechanisms are akin to inflammatory processes. Solid tumours, due to their abnormal vasculature, are often hypoxic and show increased levels of adenosine that may be an important mediator of tumour-associated immunosuppression. It is likely that killer T cells that may be recruited against cancer cells fail to act in an effective manner due to the high level of adenosine in the tumour microenvironment. Because several of these immunosuppressive effects have been attributed to the stimulation of A3 and A2A receptors, expressed on the surface of T cells, adenosine antagonists of these subtypes may be potentially useful in the immunotherapy of cancer. The interest in the elucidation of A3 adenosine receptor involvement in inflammation is evident from the large amount of experimental work carried out in peripheral blood cells of the immune system and in a variety of inflammatory conditions. A3 adenosine receptor subtype play a complex role as both pro and anti-inflammatory effects depending not only on the cell types investigated but also on the model of inflammation used and the species considered. In this study we discuss developments in our understanding of the role of adenosine A3 receptor activation in the function of the different types of cells of the immune system including neutrophils, eosinophils, lymphocytes, monocytes, macrophages and dendritic cells. Then we focused our attention on the role of adenosine in atherosclerosis, a chronic inflammatory disease of the arteries, characterized by an hypoxic region with an high concentration of adenosine and a large number of foam cells. Foam cells formation by oxidized low-density lipoprotein (oxLDL) accumulation in macrophages is crucial for development of atherosclerosis. Hypoxia has been demonstrated in atherosclerosis and hypoxia-inducible factor-1 (HIF-1) has been shown to promote intraplaque angiogenesis and foam cells development. As hypoxia induces HIF-1α stabilization and adenosine accumulation, we investigated whether this nucleoside regulates HIF-1α in FC. Adenosine, under hypoxia, stimulates HIF-1α accumulation by activating all adenosine receptors, while it has only a slight effect in normoxia. HIF-1α modulation involved extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (Akt) phosphorylation in the case of A1, A2A, A2B and ERK 1/2 phosphorylation in the case of A3 receptors. Ado, through the activation of A3 and A2B receptors, stimulates vascular endothelial growth factor (VEGF) secretion in a HIF-1α dependent way. Furthermore ado, through the A2B subtype, induces an increase of Interleukin-8 (IL-8) secretion in a ERK 1/2, p38 and Akt kinases-dependent but not HIF-1α-mediated way. Finally, adenosine stimulates FC formation and this effect is strongly reduced by A3 and A2B blockers and by HIF-1α silencing. In conclusion this study provides the first evidence that A3, A2B or mixed A3/A2B antagonists may be useful to block important steps in the atherosclerotic plaque development adoinduced.

L’artrite reumatoide, la spondilite anchilosante e l’artrite psoriasica sono malattie infiammatorie croniche, progressive e invalidanti che colpiscono le articolazioni provocando dolore e disabilità. I recettori dell’adenosina giocano un ruolo fondamentale nel meccanismo infiammatorio, in particolare l’attivazione dei sottotipi recettoriali A2A e A3 è spesso associata ad una riduzione dello stato infiammatorio. Il primo obiettivo di questo studio è stato quello di indagare il coinvolgimento dei recettori adenosinici nei pazienti affetti da artrite reumatoide all’esordio della patologia (non ancora in cura), artrite reumatoide, spondilite anchilosante ed artrite psoriasica. L’analisi dell’RNA messaggero (mRNA) e gli esperimenti di saturazione del binding hanno indicato una sovraespressione dei recettori A2A e A3 dell’adenosina nei linfociti ottenuti dai pazienti, comparati con soggetti di controllo sani. Gli agonisti dei recettori adenosinici A2A e A3 sono stati in grado di inibire l’attivazione di NF-κB, un complesso proteico funzionante come fattore di trascrizione. Inoltre hanno ridotto il rilascio di citochine pro infiammatorie, come ad esempio TNF-α, IL-1β e IL-6. Per di più l’attivazione dei sottotipi recettoriali A2A e A3 è stata in grado di mediare una riduzione delle metalloproteasi (MMP)-1 e MMP-3. L’effetto degli agonisti è stato annullato grazie alla somministrazione di antagonisti recettoriali selettivi, dimostrando così il diretto coinvolgimento di questi sottotipi recettoriali. Questi dati confermano l’implicazione dei recettori dell’adenosina nelle patologie reumatiche cronico degenerative evidenziando la possibilità di utilizzare i recettori A2A e A3 dell’adenosina come target terapeutici, con lo scopo di limitare la risposta infiammatoria spesso associata ad artrite reumatoide, spondilite anchilosante ed artrite psoriasica. Lo scopo del secondo capitolo di questa tesi, è stato quello di valutare la modulazione dei recettori A2A e A3 dell’adenosina nei pazienti affetti dalle patologie prese in esame nel primo capitolo, dopo diversi trattamenti farmacologici. Abbiamo indagato sulla densità e la funzionalità recettoriale nella progressione delle patologie attraverso uno studio longitudinale nei pazienti affetti da artrite reumatoide, spondilite anchilosante ed artrite psoriasica prima e dopo le terapie in uso, quali metotressato, agenti anti-TNFα o rituximab. I recettori A2A e A3 dell’adenosina sono stati analizzati attraverso esperimenti di saturazione del binding nei linfociti dei pazienti presi in esame, durante un periodo di ricerca della durata di 24 mesi. Nei linfociti ottenuti dai pazienti affetti da artrite reumatoide, la sovraespressione dei sottotipi recettoriali A2A e A3 dell’adenosina è stata gradualmente ridotta in funzione del tempo di trattamento. Questi risultati confermano il coinvolgimento dei recettori adenosinici A2A e A3 nella progressione delle patologie reumatiche cronico degenerative, sottolineando che gli agonisti dei recettori A2A e A3 dell’adenosina potrebbero rappresentare un’alternativa terapeutica per il trattamento dell’artrite reumatoide.
Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A2A and A3AR subtypes is often associated with a reduction of the inflammatory status. The first aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A2A and A3ARs in lymphocytes obtained from patients when compared with healthy subjects. A2A and A3AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Moreover, A2A and A3AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. These data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A2A and A3ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA. The purpose of the second chapter of this thesis, was to evaluate the modulation of A2A and A3ARs in patients suffering from RA, AS and PsA after different pharmacological treatments. We investigated A2A and A3AR density and functionality in pathologies progression by using a longitudinal study in RA, AS and PsA patients before and after methotrexate (MTX), anti-TNFa agents or rituximab treatments. A2A and A3ARs were analyzed by saturation binding assays in lymphocytes from patients throughout the 24-month study timeframe. In lymphocytes obtained from RA patients, the A2A and A3AR up-regulation was gradually reduced in function of the treatment time. Taken together, these data confirmed the involvement of A2A and A3ARs in chronic inflammatory rheumatic disease progression and highlighted that A2A and A3AR agonists could represent a physiological-like therapeutic alternative for RA treatment.

Master of Science
Department of Kinesiology
Brett J. Wong
Mechanisms underlying the cutaneous vasodilation response to local skin heating and whole body heating in humans remain unresolved. Although nitric oxide (NO) is known to contribute to these responses, it remains unclear as to the source of NO. Adenosine receptors induce vasodilation in many human tissues and may work, in part, through NO. As these receptors are also known to be located in the cutaneous vasculature, the studies contained in this thesis were designed to investigate a potential contribution of adenosine receptor activation to the rise in skin blood flow elicited by local skin and whole body heating. The study presented in chapter IV was designed to determine a potential role for adenosine receptors in contributing to cutaneous thermal hyperemia. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments designed to elucidate the contribution of A[subscript]1/A[subscript]2 adenosine receptors during local skin heating. Each site was locally heated from a baseline temperature of 33°C to 42°C at a rate of 1°C/10 s and skin blood flow was monitored via laser-Doppler flowmetry (LDF). The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation directly contributes to cutaneous thermal hyperemia. These data further suggest a portion of the NO response may be explained by A[subscript]1/A[subscript]2 adenosine receptor activation; however, a substantial portion of the NO response is independent of the adenosine receptor contribution. The study presented in chapter V was designed to determine a potential role for A[subscript]1/A[subscript]2 adenosine receptors in contributing to cutaneous active vasodilation. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments, as above, and skin blood flow was monitored via LDF. Whole body heat stress, sufficient to raise oral temperature at least 0.8°C above baseline, was induced via water-perfused suits. The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation does not directly contribute to cutaneous active vasodilation; however, a role for A[subscript]1/A[subscript]2 adenosine receptor activation is unmasked when NO synthase is inhibited. The data from this study further suggest that A[subscript]1/A[subscript]2 adenosine receptor activation may be responsible for a portion of the known NO component of cutaneous active vasodilation.

Adenosine (Ado) is an endogenous nucleoside ubiquitous in mammals promoting protection and cells repair during metabolic stress conditions. Through interaction with the four Ado receptor subtypes (ARs), AR ligands have shown potential therapeutic interest for many disorders. In this work both new A2AAR agonists and A3AR antagonists were designed, synthesized and tested in vitro. Although  Ado  5’-N-ethylcarboxamide derivatives like VT 7 and GCS21680 display a good affinity and selectivity for A2AAR, the development of new agonists for this receptor subtype is still a big challenge in nucleoside chemistry. In this current decade, some papers have reported  that  a  tetrazolyl  residue  in  4’-position of Ado derivatives led to compounds endowed with good A2AAR affinity. Hence, in this work, compounds bearing the Nethyltetrazoyl  moiety  in  4’- position of the Ado ribose portion together with different arylalkylthio and arylalkylamino chains in C2-position were designed and synthesized. The new compounds were prepared using a convergent approach. To this purpose, 2,6- dichloropurine was coupled with the suitable modified sugar to afford a nucleoside which was further modified by introducing an amino group at the C6- and the suitable side chain at the C2- position. The modified sugar used in the coupling reaction was synthesized starting from the commercially available D-ribose in seven steps. The binding assay and functional study performed with the new compounds at all AR subtypes transfected on Chinese hamster ovary (CHO) cells revealed that the 2-phenylethylthio derivative was the compound endowed with the better affinity for the A2AAR/A3AR subtypes (17: Ki hA2AR = 5.8 nM; Ki hA3R = 1.2 nM). It is worthwhile to note that the presence of the ethyltetrazolyl substituent in the sugar moiety favors the interaction with the receptor respect to the ethylcarboxamido group. As expected, the new derivatives show a dual behavior at ARs, resulting A2AAR agonists and A3AR antagonists (17: IC50 at hA3AR of 8.4 nM). Furthermore, the wound healing potential of the news nucleosides was evaluated respect to VT 7, CGS21680 and epidermal growth factor (EGF, used as positive control). The compounds, 17, 19, 20, 21 and 22 showed all better wound healing potential respect to VT 7, CGS21680 and EGF. Therefore such compounds are good candidates for further investigation in in vivo model of wound healing. New A3AR antagonists were also prepared based on the observations that, the substitution of the 8-bromine atom of 8-bromo-9-ethyladenine (Ki hA2AAR = 52 nM, Ki hA3AR = 2,800 nM) with phenylacetylene shifts the preference of the resulting compound (Ki hA3AAR = 86 nM, hA2AAR= 600 nM) from A2AAR to A3AR. Hence, from these facts, three series of compounds were prepared. The first one was 8-phenylethynyladenine derivatives substituted at N-9 position with different alkyl/arylalkyl chains. The second one combines substitution on the phenyl ring of 8- phenylethynyladenine with either N-9 cyclopentyl or N9 phenethyl since they resulted being the best N-9 substituents of the first series. The third series combines a fixed para-methoxy- phenylacetylene in C-8 with either N-9 cyclopentyl or N-9 phenethyl, 2-chloro, and with different N6 substituents. The 8-arylethynyladenine derivatives substituted at 9 position with different alkyl/arylalkyl chains, and the corresponding compounds further substituted at the 2 and N6 position, were synthesized starting from commercially available adenine or 2,6- dichlorpurine in three/five steps, respectively. The results of the in vitro test reported that: N-9 cyclopentyl improved affinity while N-9 phenylethyl improved selectivity, the chlorine atom is well tolerated especially when combined with C-8 para-methoxy-phenylacetylene, the N6 substitution gave compounds with maintained selectivity in the same range of the non- substituted derivatives but with a slight decrease of the affinity. Most of the new compounds are endowed with high affinity and different degree of selectivity for the A3AR subtype. In particular, the tetrasubstituted adenine derivative 38 (Ki A3R = 8.4 nM; Ki A1R and Ki A2AR >30,000 nM) resulting the most active and selective ligand and it represents a very good ligand to study the A3AR subtype and its function.

The aim of this work was the design and synthesis of new A2A and A3 adenosine receptors antagonists. Two different studies have been performed. The first one based on structural modifications of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pirimidine nucleus, reported in literature. In order to identify a new series of A2A or A3 AR antagonists and with the aim to better investigate the role of the nitrogen at the 7- position on the interaction with ARs, it was performed a synthetic strategy for the preparation of the pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus which can be considered the 7-deaza-analogue of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine core. In order to complete the SAR studies on this class of compounds, it has been synthesised a novel series of pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives which can be considered the structural isomers of the parent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. From the biological data obtained, we can assert that the concomitant presence of the N7 and N8 is fundamental for the selectivity of these A2A/A3 ligands versus the remaining ARs subtype. Our results confirmed the importance of the presence of the NH at the 5- position of the PTPs nuclus, this could be due to the formation of an essential ligand-receptor hydrogen bond. Most of these compounds, revealed to be non selective ARs antagonists. Derivative 5-{[(4-methoxy-phenyl)carbamoyl]amino}-(2-furan-2-yl)-8-methyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine resulted to be the best compound of the series in term of both affinity (hA3Ki = 15nM) and selectivity. The aim of the second project was to obtain A3 antagonists with improve water-solubility. A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/ethyl-2-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-one derivatives have been synthesised and evaluated in radioligand binding assays to determine their affinities at the human A1, A2A, and A3 adenosine receptors. Efficacy at the hA2B AR and antagonism of selected ligands at the hA3 AR were also assessed through cAMP experiments. All the synthesised molecules exhibited high affinity at the hA3 AR (Ki values ranging from 1.46 to 44.8 nM) as well as remarkable selectivity versus A1, A2A and A2B AR subtypes. In view of the chirality of the tricycles, for selected compounds both the racemic mixtures and the pure enantiomers have been prepared with the purpose of determine a possible receptor stereoselectivity. Compound (R)-4-allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one showed to be the most potent hA3 AR ligand of the series (hA3Ki = 1.46 nM), in addition with very high selectivity over all the other ARs (hA2AKi/ hA3Ki > 3425; hA2BIC50/ hA3Ki > 3425; hA1Ki/ hA3Ki = 1,729).

El desenvolupament del sistema nerviós perifèric implica una inicial exuberant producció de neurones i, una posterior reducció dependent de l'activitat del nombre de sinapsis de les unions neuromusculars (NMJ). Aquest procés s’anomena eliminació sinàptica. Al final de la primera setmana postnatal, cada fibra muscular està innervada per una sola motoneurona. Els receptors muscarínics d’acetilcolina (mAChR), els receptors d’adenosina (AR) i el receptor cinasa de tropomiosina B (TrkB) podrien permetre la competició entre terminals nerviosos durant el procés d’eliminació sinàptica mitjançant la modulació de l’alliberament d’acetilcolina. En aquesta tesi s’ha investigat, mitjançant microscòpia confocal i un anàlisi morfològic quantitatiu, el paper dels receptors mAChRs (M1, M2 i M4), dels AR (A1 i A2A) i del receptor TrkB en el procés d’eliminació en el desenvolupament de la NMJ. Els resultats mostren que els receptors mAChRs, AR i el receptor TrkB promouen una desconnexió axonal al principi de la segona setmana postnatal independentment de la maduració dels receptors d’acetilcolina postsinàptics. En resum, els receptors mAChRs, AR i el receptor TrkB endarrereixen el procés d’eliminació sinàptica a P7 però l’acceleren a P9. Pel que fa la cooperació d’aquests receptors, M4 produeix un efecte oclusiu sobre M1 i un efecte additiu sobre a P7. La cooperació entre els receptors M1, A1 i A2A promou la pèrdua axonal a P9, mentre que, l’efecte de M2 és independent dels altres receptors. El M1 i TrkB treballen junts per incrementar la pèrdua axonal a P9 independentment dels receptors M2 i TrkB. En conclusió, l’eliminació sinàptica postnatal és regulada per un mecanisme que depèn de varis receptors, involucrant la cooperació dels diferents subtipus de receptors muscarínics, d’adenosina i el receptor TrkB, els quals garanteixen la monoinnervació de les sinapsis neuromusculars al final del procés.
El desarrollo del sistema nervioso periférico implica una inicial exuberante producción de neuronas y, una posterior reducción dependiente de actividad del número de sinapsis en las uniones neuromusculares (NMJ). Este proceso se denomina eliminación sináptica. Al final de la segunda semana postnatal, cada fibra muscular esta inervadas por una solo motoneurona. Los receptores muscarínicos de acetilcolina (mAChR), los receptores de adenosina (AR) y el receptor quinasa de tropomiosina B (TrkB) podrían permitir la competición entre los terminales nerviosos durante el proceso de eliminación sináptica mediante la modulación en la liberación de acetilcolina. En esta tesis se ha investigado, mediante microscopía confocal y un análisis morfológico cuantitativo, el papel de los receptores mAChRs (M1, M2 y M4), de los receptores de adenosina (A1 y A2A) y del receptor TrkB en el del proceso de eliminación en el desarrollo de la NMJ. Los resultados muestran que los receptores mAChRs, AR y el receptor TrkB promueven una desconexión axonal al inicio de la segunda semana postnatal independientemente de la maduración de los receptores de acetilcolina postsinápticos. En resumen, los receptores mAChRs, AR y el receptor TrkB retrasan el proceso de eliminación sináptica en P7 pero lo aceleran en P9. En la cooperación de estos receptores, se ha demostrado que M4 produce un efecto oclusivo sobre M1 y aditivo sobre A1 en P7. La cooperación entre M1, A1 y A2A promueve la pérdida axonal en P9, mientras que M2 es independiente de los otros receptores. M1 y TrkB cooperan para incrementar la pérdida axonal en P9 independientemente de M2 y TrkB. En conclusión, la eliminación sináptica postnatal está regulada por un mecanismo que depende de varios receptores, involucrando la cooperación de diferentes subtipos de receptores muscarínicos, de adenosina y el receptor TrkB, los cuales garantizan la monoinnervación de las sinapsis neuromusculares al final del proceso. saludable.
The development of the peripheral nervous system involves an initially exuberant production of neurons and a subsequent activity-dependent reduction in the number of synapses at the neuromuscular junctions (NMJ). This process is called synaptic elimination. At the end of the first postnatal week, each muscle fiber is innervated by a single motoneuron. Muscarinic acetylcholine receptors (mAChR), adenosine receptors (AR) and the tropomyosin-related kinase B (TrkB) receptor may allow the direct competition between nerve endings during synapse elimination through the modulation of acetylcholine release. Here, it has been investigated by confocal microscopy and quantitative morphological analysis the involvement of the individual and synergic or oclusive effect of M1-, M2- and M4-subtypes of mAChRs, A1 and A2A of ARs and TrkB in the control of the axonal elimination in developing NMJ. The results show that mAChRs, ARs and TrkB promote axonal disconnection at the beginning of the second postnatal week without affecting the postsynaptic maturation of the nicotinic receptor cluster. In summary, mAChRs, ARs and TrkB delay axonal loss at P7 but accelerate it at P9. In terms of receptor cooperation, M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway at P7. The cooperation between M1, A1 and A2A receptors promotes axonal loss at P9, whereas the effect of M2 is independent of the other receptors. M1 and TrkB receptors work together to increase axonal loss rate at P9 but the effect of M2 is largely independent of the TrkB receptor. In conclusion, postnatal synapse elimination is a regulated multireceptor mechanism involving the cooperation of several muscarinic, adenosine and TrkB receptor subtypes that guarantees the monoinnervation of the neuromuscular synapses in the end of the process.

2016 - 2017
The adenosinergic pathway plays a critical role in cancer development and progression, as well as in drug resistance to chemotherapy and/or targeted-therapy. The goal of this PhD thesis was to investigate and fully characterize the role of CD73/adenosine A2A-A2B receptors axis in cancer, highlighting the therapeutic potential of inhibitors of the adenosinergic pathway. We firstly characterized the mechanism/s by which A2BR promotes immunosuppression and angiogenesis in tumor-bearing hosts, focusing on the role of myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs). The results revealed that treatment of melanoma-bearing mice with Bay60-6583, a selective A2BR agonist, is associated with 1. increased tumor VEGF-A expression and vessel density, and 2. increased accumulation of tumor-infiltrating CD11b+Gr1+cells (MDSCs). MDSCs strongly contribute to the immunosuppressive and angiogenic effects of Bay60-6583. Melanoma-bearing mice treated with a selective A2BR antagonist PSB1115 showed reduced tumor growth compared to controls and this effect was associated with reduced tumor angiogenesis, low levels of MDSCs and increased number of tumor-infiltrating CD8+ T cells. Furthermore, blockade of A2BR increased the anti-tumor effects of VEGF-A inhibitors. Next, we verified that A2BR activation also drives fibroblasts activation within melanoma tissues, by increasing the number of FAP positive cells within tumor lesions. FAP is a common marker of activated fibroblasts also named cancer-associated fibroblasts. These cells produce and secrete various tumor-promoting factors, including fibroblast growth factor (FGF)-2 and CXCL12 or stromal-derived factor 1 α (SDF1α), that were increased both in melanoma tissue and fibroblasts isolated from melanoma tissue or from skin upon Bay60-6583 treatment. Bay60-6583-induced FGF-2 from fibroblasts contributed to melanoma cells proliferation. The CXCL12/CXCR4 pathway, instead, was involved in the pro-angiogenic effects of A2BR agonist, but not in its immunosuppressive effects. These effects were significantly blocked by the A2BR antagonists PSB1115. Taken together, these data elucidate the pivotal role of A2BR in establishing a positive cross-talk between tumor-infiltrating immune cells, fibroblasts and endothelial cells that sustain tumor growth, reinforcing the therapeutic potential of A2BR blockers for cancer therapy. ... [edited by Author]
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Il segno distintivo del processo neuroinfiammatorio è l'attivazione delle microglia, le cellule immunocompetenti del sistema nervoso centrale (SNC), che sono responsabili del rilascio di numerosi mediatori pro-infiammatori implicati nella patogenesi di diverse malattie neurologiche come la malattia di Alzheimer, il morbo di Parkinson, la sclerosi multipla e l’ictus ischemico. Il recettore dell’immunità innata Toll-like (TLR)4, presente sulla superficie della microglia, gioca un ruolo chiave nella difesa dell'organismo e può essere attivato dal lipopolisaccaride (LPS), uno dei componenti della membrana cellulare esterna dei batteri gram-negativi. I recettori dell'adenosina (AR) interagendo con il recettore TLR4 influenzano numerose proprietà immunologiche delle cellule microgliali e sono implicati in molteplici stati fisiologici e patofisiologici nel SNC. Infatti, è stato dimostrato che l'adenosina (Ado), nucleoside ubiquitario con importanti funzioni immunomodulatorie e ligando endogeno degli AR, prende parte ai principali processi di attivazione della microglia come: proliferazione, estensione e retrazione dei processi cellulari, migrazione e produzione di citochine. Sulla base di queste considerazioni, lo scopo di questa tesi è stato quello di investigare il contributo adenosinergico sulla neuroinfiammazione mediata dalle cellule gliali attraverso l’analisi dell'espressione degli AR e le vie di trasduzione del segnale, i fattori di trascrizione e le citochine attivate da questi recettori in diverse condizioni patofisiologiche legate ad ipossia e infiammazione. Lo scopo dello studio riportato nel capitolo 1 è stato quello di investigare se Ado poteva influenzare le funzioni della microglia agendo sulla modulazione del fattore indotto dall’ipossia-1α (HIF-1α), il principale fattore di trascrizione dei geni ipossia-inducibili, coinvolti nella risposta immunitaria e regolati in normossia da mediatori dell'infiammazione. I dati ottenuti hanno messo in evidenza che Ado è in grado di aumentare l'accumulo di HIF-1α indotto dall’LPS portando ad un aumento dei geni bersaglio di HIF-1α coinvolti nel metabolismo cellulare e nell’eliminazione dei patogeni ma non è in grado di indurre quelli dipendenti da HIF-1α correlati all'angiogenesi e all'infiammazione. L'effetto stimolatorio di Ado su HIF-1α e dei suoi geni bersaglio è stato essenzialmente esercitato dall'attivazione degli A2A AR attraverso le proteine chinasi attivate da mitogeni (MAPK) chinasi regolate da segnali extra cellulari (ERK)1/2 e degli A2B AR tramite la MAPK p38 e la fosforilazione di Akt. Inoltre, Ado attraverso gli A2B AR ha aumentato i livelli del fattore di crescita dell'endotelio vascolare (VEGF) e diminuito quelli del fattore di necrosi tumorale (TNF)-α. Lo scopo dello studio riportato nel capitolo 2 è stato quello di indagare il potenziale ruolo degli AR nella modulazione della secrezione dell'interleuchina(IL)-6 e nella proliferazione cellulare in cellule microgliali murine primarie. I dati ottenuti hanno messo in evidenza che l'agonista A2B (BAY 60-6583) è in grado di stimolare l'aumento di IL-6, in modo dose e tempo-dipendente, sia in normossia che in ipossia e che tale aumento è fortemente ridotto dall’incubazione delle cellule con gli inibitori della fosfolipasi C (PLC), della proteina chinasi C (PKC)-ε e PKC-delta. Lo studio sulle vie di trasduzione del segnale cellulare ha rivelato che solo l'inibitore di p38 è in grado di bloccare l'effetto dell’agonista A2B sulla secrezione di IL-6, mentre gli inibitori di ERK1/2, JNK1/2 e Akt non hanno dato alcun effetto. La stimolazione di p38 da parte di BAY 60-6583 è risultata dipendente dagli A2B AR, tramite PLC, PKC-ɛ e PKC-delta ma non tramite l’adenilato ciclasi, sia in normossia che in ipossia. Infine, BAY 60-6583 ha aumentato la proliferazione delle cellule microgliali attraverso la via di segnale che vede coinvolti A2B AR, PLC, PKC-ε PKC-delta e p38.
The hallmark of neuroinflammation is the activation of microglia, the immunocompetent cells of the CNS, releasing a number of pro-inflammatory mediators implicated in the pathogenesis of several neurological diseases such as AD, PD, MS and ischemic stroke. The innate immune TLR4, localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms and LPS, a component of the cell wall of gram-negative bacteria, was first identified as the TLR ligand. ARs interacting with TLR4 influence on many immune properties of microglia and are implicated in numerous physiological and pathophysiological states in CNS. Indeed, Ado, a ubiquitous nucleoside with important immunomodulatory functions, has been found to take part in the principal microglial activation processes spanning from proliferation, process extension, retraction, migration and cytokine production. On this background, the aim of this study was to investigate the adenosinergic contribution to glial cell mediated-neuroinflammation by analyzing the expression of ARs and the signalling pathways, transcription factors and cytokines activated by them in different pathophysiological conditions linked to hypoxic and inflammatory conditions. The aim of studies reported in chapter 1 was to investigate whether Ado may affect microglia functions by acting on HIF-1α modulation, the main transcription factor of hypoxia-inducible genes, involved in the immune response, being regulated in normoxia by inflammatory mediators. Primary murine microglia were activated with LPS with or without Ado, Ado receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Ado increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism and pathogens killing but did not induce HIF-1α dependent genes related to angiogenesis and inflammation. The stimulatory effect of Ado on HIF-1α and its target genes was essentially exerted by activation of A2A through ERK1/2 (or p44/42) and A2B subtypes via p38 MAPKs and Akt phosphorylation. Furthermore, the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. Our results show that Ado increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B ARs, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. The aim of studies reported in chapter 2 was to indagate the potential role of ARs in the modulation of IL-6 secretion and cell proliferation in primary microglial cells. The A2B AR agonist BAY 60-6583 stimulated IL-6 increase under normoxia and hypoxia, in a dose- and time-dependent way. In cells incubated with the blockers of PLC, PKC-ε and PKC-delta the IL-6 increase due to A2B AR activation was strongly reduced, whilst it was not affected by the inhibitor of AC. Investigation of cellular signalling involved in the A2B AR effect revealed that only the inhibitor of p38 MAPK was able to block the agonist effect on IL-6 secretion, whilst inhibitors of ERK1/2, JNK1/2 MAPKs and Akt were not. Stimulation of p38 by BAY 60-6583 was A2B AR dependent, through PLC, PKC-ɛ and PKC-delta but not AC pathway, in both normoxia and hypoxia. Finally, BAY 60-6583 increased microglial cell proliferation involving A2B AR, PLC, PKC-ε PKC-delta and p38 signalling. Our results show for the first time that Ado by activating A2B ARs increases IL-6 protein levels and cell proliferation through a pathway dependent on PLC, PKC-ε, PKC-delta, and p38 signalling. These findings add new molecular pathways activated by Ado in microglia to give a reduction of genes and cytokines involved in inflammation and hypoxic injury that may cohexist in stroke, ischemia and other CNS disorders.

Chapter 1 of thesis is a literature review of adenosine research. The central importance of the contributions of both classical pharmacology and, more recently, molecular biology to adenosine research is demonstrated. These disciplines have enabled the classification and characterisation of adenosine receptors and as well an understanding of the physiological significance of endogenous adenosine. The significant benefits of developing therapeutics for regulation of the diverse physiological functions of adenosine, by regulation of adenosine receptors, is outlined. For this therapeutic potential to be realised both high affinity and subtype selective adenosine agonists and antagonists are required. The structure-activity relationships for agonists and xanthine antagonists are discussed. The assimilation of these structure-activity relationships have guided the development of ligand based models of the adenosine receptor pharmacophore. The 'flipped', 'N6-C8' and 'three binding domain' models were described. These models aim to direct the future design of high affinity and selective ligands for adenosine receptors. The development of receptor based models by modelling of the receptor-ligand complex is also presented. The main body of this thesis presents a study of the structure-activity relationships for pyrazolo(3,4-d) pyrimidines binding to adenosine Ai and A2a receptors. Prior to this study few non-xanthine adenosine antagonists had been well defined or optimised in terms of structure-activity relationships. However, the value of such ligands is immense, facilitating further definition of structural requirements for high affinity and selective adenosine receptor binding. These ligands should complement existing agonists and xanthine antagonists in developing an understanding of adenosine receptor binding. The experimental approach to development of the lead compound of this study, a-(6-(l'-carbamoylethylthio)- l-phenylpyrazolo(3,4-d)pyrimidin-4-ylthio)propanamide (5), is outlined in Chapter 2 of this thesis. 5 is substituted at C-4, C-6 and N-i of the pyrazolo(3,4-d)pyrimidine heterocycle. The experimental approach to optiniising 5 was approached in a rational manner, requiring an iterative approach i.e. design of generation I target compounds --synthesis -- biological evaluation -- structure-activity relationships -- design of generation II target compounds, etc. Chapters 3, 4 and 5 of this thesis describe this experimental approach as it relates to optimising the lead compound, 5, for adenosine receptor affinity and subtype selectivity. The importance of receptor interactions with multiple ligand domains, to achieve both potency and selectivity, was recognised so that optimisation of the C-4, C-6 and N-i substituents of the lead compound was targeted and achieved. Previous structure-activity studies with agonists and xanthine antagonists have concentrated on modifying a single ligand domain. Chapter 3 presents twelve generation I target compounds to examine C-4 and C-6 substituent structure-activity relationships. Chapter 4 presents twelve generation II target compounds to further examine C-4 and C-6 substituent structure-activity relationships. Chapter 5 presents sixteen generation ifi target compounds to examine N-I substituent structure-activity relationships. A major outcome from the research presented in these chapters was the development of highly potent and highly selective ligands for the adenosine A1 receptor subtype. a(4-Methylamino- I -phenylpyrazolo(3,4-d)pyrimidin-6-ylthio)hexanamide (29) was the most potent ligand at the Ai receptor identified in this study, and is one of the most potent Ai selective antagonists ever reported. 29 has an A1 K1 value of 0.745±0.045 nM and is 332-fold selective for the A1 receptor over the A2a receptor. a-(1-Phenyl-4-propylthiopyrazolo(3,4-d)pyrimidin-6-ylthio)butanainide (27) was the most selective ligand of this study. It is four orders of magnitude selective for the A1 receptor (up to 16900-fold), and one of the most selective antagonists ever reported. This high selectivity has been achieved with the maintenance of good A1 affinity (A1 K1 = 29.5±6.6 nM). These results prove the value of modifying multiple substituents of adenosine receptor ligands, generating ligands which bind with high potency and selectivity to adenosine Al receptors compared to adenosine A2a receptors.

Chapter 1 of thesis is a literature review of adenosine research. The central importance of the contributions of both classical pharmacology and, more recently, molecular biology to adenosine research is demonstrated. These disciplines have enabled the classification and characterisation of adenosine receptors and as well an understanding of the physiological significance of endogenous adenosine. The significant benefits of developing therapeutics for regulation of the diverse physiological functions of adenosine, by regulation of adenosine receptors, is outlined. For this therapeutic potential to be realised both high affinity and subtype selective adenosine agonists and antagonists are required. The structure-activity relationships for agonists and xanthine antagonists are discussed. The assimilation of these structure-activity relationships have guided the development of ligand based models of the adenosine receptor pharmacophore. The 'flipped', 'N6-C8' and 'three binding domain' models were described. These models aim to direct the future design of high affinity and selective ligands for adenosine receptors. The development of receptor based models by modelling of the receptor-ligand complex is also presented. The main body of this thesis presents a study of the structure-activity relationships for pyrazolo(3,4-d) pyrimidines binding to adenosine Ai and A2a receptors. Prior to this study few non-xanthine adenosine antagonists had been well defined or optimised in terms of structure-activity relationships. However, the value of such ligands is immense, facilitating further definition of structural requirements for high affinity and selective adenosine receptor binding. These ligands should complement existing agonists and xanthine antagonists in developing an understanding of adenosine receptor binding. The experimental approach to development of the lead compound of this study, a-(6-(l'-carbamoylethylthio)- l-phenylpyrazolo(3,4-d)pyrimidin-4-ylthio)propanamide (5), is outlined in Chapter 2 of this thesis. 5 is substituted at C-4, C-6 and N-i of the pyrazolo(3,4-d)pyrimidine heterocycle. The experimental approach to optiniising 5 was approached in a rational manner, requiring an iterative approach i.e. design of generation I target compounds --synthesis -- biological evaluation -- structure-activity relationships -- design of generation II target compounds, etc. Chapters 3, 4 and 5 of this thesis describe this experimental approach as it relates to optimising the lead compound, 5, for adenosine receptor affinity and subtype selectivity. The importance of receptor interactions with multiple ligand domains, to achieve both potency and selectivity, was recognised so that optimisation of the C-4, C-6 and N-i substituents of the lead compound was targeted and achieved. Previous structure-activity studies with agonists and xanthine antagonists have concentrated on modifying a single ligand domain. Chapter 3 presents twelve generation I target compounds to examine C-4 and C-6 substituent structure-activity relationships. Chapter 4 presents twelve generation II target compounds to further examine C-4 and C-6 substituent structure-activity relationships. Chapter 5 presents sixteen generation ifi target compounds to examine N-I substituent structure-activity relationships. A major outcome from the research presented in these chapters was the development of highly potent and highly selective ligands for the adenosine A1 receptor subtype. a(4-Methylamino- I -phenylpyrazolo(3,4-d)pyrimidin-6-ylthio)hexanamide (29) was the most potent ligand at the Ai receptor identified in this study, and is one of the most potent Ai selective antagonists ever reported. 29 has an A1 K1 value of 0.745±0.045 nM and is 332-fold selective for the A1 receptor over the A2a receptor. a-(1-Phenyl-4-propylthiopyrazolo(3,4-d)pyrimidin-6-ylthio)butanainide (27) was the most selective ligand of this study. It is four orders of magnitude selective for the A1 receptor (up to 16900-fold), and one of the most selective antagonists ever reported. This high selectivity has been achieved with the maintenance of good A1 affinity (A1 K1 = 29.5±6.6 nM). These results prove the value of modifying multiple substituents of adenosine receptor ligands, generating ligands which bind with high potency and selectivity to adenosine Al receptors compared to adenosine A2a receptors.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
Science, Environment, Engineering and Technology
Full Text

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Adenosine is a neuromodulator able to control the balance between neuronal excitation and inhibition, throughout the central nervous system (CNS). Adenosine acts onto a G protein–coupled receptors (GPCRs) subfamily called P1 adenosine receptors: A1R, A2AR, A2BR and A3R. Adenosine type 2A receptor (A2AR) is highly expressed in striatum, whereas in other brain regions A2AR is expressed at lower levels. One of these regions is the hippocampus, a central brain region enrolled in learning and memory processes as well as spatial recognition. Moreover, it was discovered that A2AR modulate reference memory. Additionally, A2AR can facilitate synaptic transmission since it can fine-tune other neuromodulatory systems, by controlling neurotransmitter release and modulating metabotropic or ionotropic receptors. It was also described that A2AR can interact with NMDA receptor (NMDAR), that is an ionotropic receptor. Therefore, given the particular importance of NMDAR in the hippocampal neurotransmission phenomena, in this work, we explore how A2AR can modulate NMDAR-dependent currents in hippocampal Schaffer collaterals - CA1 synapses. We showed that exogenous activation of A2AR, with its selective agonist CGS21680, decreases NMDAR-dependent evoked excitatory postsynaptic currents (eEPSCs) and that this effect is no longer observed in the presence of the selective A2AR antagonist, SCH58261. However, the superfusion of SCH58261 alone also decreases NMDAR-eEPSCs, suggesting that A2AR may exert a tonic control of these currents in CA1 pyramidal cells. Finally, this study revealed that our slice preparations contained high levels of endogenous adenosine which, once removed, does not prevent the A2AR antagonist-induced decrease in NMDAR-eEPCSs in SC-CA1 synapses. Our main hypothesis to explain these results is based in the possibility of existing two sub-populations of A2AR that may have antagonistic effects upon NMDA-dependent currents. Nevertheless, this hypothesis and mechanisms remain to be clarified, which can be the foundation for future work.
A adenosina é um neuromodulador capaz de controlar o balanço entre excitação e inibição ao longo de todo o sistema nervoso central. Este controlo é feito através da activação da subfamília de receptores acoplados a proteínas G (GPCRs), os receptores de adenosina P1: A1R, A2AR, A2BR and A3R. O receptor de adenosina do tipo 2A (A2AR) é abundantemente expresso no estriado e é menos expresso noutras regiões do cérebro. Uma destas regiões é o hippocampo, uma região cerebral central envolvida nos processos de aprendizagem, memória e reconhecimento espacial. Foi descoberto que os A2AR podem modular a memória de referência. Além disso, os A2AR estão maioritariamente descritos como facilitadores da transmissão sináptica, sendo que as suas principais funções são o refinamento de outros sistemas neuromodulatórios, o controlo da libertação de neurotransmissores e ainda a modulação da actividade de outros receptores, quer metabotrópicos quer ionotrópicos.O receptor de NMDA (NMDAR) é um receptor ionotrópico com o qual o A2AR pode interagir. Deste modo, dada a particular importância do NMDAR para o fenómeno de neurotransmissão no hippocampo, este trabalho explora de que forma é que os A2AR modulam as correntes dependentes de NMDAR nas sinapses entre os neurónios piramidais do CA1 e as colaterais de Schaffer (SC) no hipocampo.Mostrou-se que a activação exógena do A2AR com o seu agonista selectivo CGS21680 diminui as correntes excitatórias pós-sinápticas evocadas (eEPSC) dependentes do receptor de NMDA, um efeito que deixa de ser observado na presença do antagonista selectivo dos A2AR, SCH58261. Além disso, SCH58261 diminui a amplitude dos eEPSCs em condições basais o que sugere que os A2AR poderão exercer um controlo tónico destas correntes nos neurónios piramidais do CA1. Finalmente, com este trabalho observou-se ainda, que as preparações in vitro utilizadas têm uma grande quantidade de adenosina endógena. A sua remoção, porém, não previne o efeito induzido pelo antagonista selectivo dos A2AR, continuando a observar-se uma diminuição na amplitude de eEPSC dependentes de NMDAR nas sinapses CA1-SC.A principal hipótese para explicar esta aparente contradição de resultados tem a ver com a possível existência de duas sub-populações de A2AR que podem afectar as correntes dependentes de NMDAR de maneira oposta. Contudo, o estudo de qual a população de A2AR que é responsável por cada um dos efeitos, assim como quais os mecanismos por detrás desta evidente modulação dos A2AR sobre os NMDAR permanece por clarificsr, o que poderá servir de basede trabalho futuro.
Outro - 016684 - PTDC/NEU-NMC/4154/2014 (POCI-01-0145-FEDER-016684) - Papel dos astrócitos no controlo da memória- foco nos recetores adenosina A2A - Fundação para a Ciência e a Tecnologia
Outro - COMPETE POCI-01-0145-FEDER-007440 - Trabalho multidisciplinar no âmbito da neurociência cognitiva na saúde e na doença - Fundação para a Ciência e a Tecnologia

Tese de Doutoramento em Ciências da Saúde - Ramo de Medicina, apresentada à Faculdade de Medicina da Universidade de Coimbra
As doenças pulmonares inflamatórias crónicas, incluindo a asma, a doença pulmonar obstrutiva crónica e doenças pulmonares intersticiais são uma causa crescente e significativa de incapacidade e mortalidade precoce à escala global. Há uma necessidade urgente e reconhecida de novas terapêuticas, mas os alvos identificados nos estudos in vitro e modelos animais não têm sido aplicados com sucesso em humanos. A modulação dos recetores purinérgicos é uma abordagem particularmente promissora no controlo da inflamação e promoção da homeostasia. Um conjunto extenso e robusto de dados pré-clínicos demonstraram a presença e atividade destes recetores em células e tecidos envolvidos na inflamação pulmonar e a sua modulação foi eficaz em modelos animais de doença. No entanto, a aplicação destes resultados em ensaios clínicos tem sido dececionante. Alguns autores argumentam que essa ineficácia resulte de diferenças na distribuição e efeitos dos recetores purinérgicos entre as espécies. O objectivo deste estudo foi a avaliação dos efeitos dos recetores da adenosina na doença pulmonar humana. Em última análise pretende-se obter uma prova de conceito na manipulação dos recetores da adenosina para o tratamento de doenças pulmonares humanas. Foi aplicada uma estratégia de três passos. Primeiro, estudaram-se os efeitos do consumo crónico de doses moderadas de cafeína, um antagonista não seletivo dos recetores A1 e A2A da adenosina, na saúde respiratória, possibilitando assim um estudo não intervencional do efeito destes recetores em humanos. De seguida, avaliaram-se os níveis pulmonares dum percursor da adenosina, permitindo a avaliação da ativação endógena dos recetores purinérgicos na doença pulmonar humana. Finalmente, desenvolveu-se um método laboratorial que avalia um marcador robusto de ativação celular em macrófagos alveolares humanos frescos e não modificados. Salienta-se que o macrófago alveolar apresenta especial importância na patogénese das doenças pulmonares inflamatórias. Os estudos epidemiológicos mostraram efeitos globalmente benéficos da cafeína nas doenças respiratórias, incluindo na asma e na tosse pós-infeciosa, bem como na função respiratória e na mortalidade de causa respiratória. Os efeitos na DPOC e sarcoidose não foram claros. Não se observou correlação entre o consumo de cafeína e o risco ou gravidade da apneia do sono. Quando se mediram os níveis pulmonares de trifosfato de adenosina (ATP), encontraram-se níveis elevados em doentes com sarcoidose, comparativamente àqueles com pneumonite de hipersensibilidade, sugerindo que o ATP pode ser útil como biomarcador auxiliar de diagnóstico. Finalmente, em macrófagos alveolares humanos, os recetores A2A da adenosina sofreram alterações adaptativas da sua densidade e a sua ativação reduziu os transientes de cálcio livre intracelular causados pela exposição a um estímulo inflamatório. Estes resultados suportam a proposta de que os recetores A2A da adenosina são um alvo promissor no tratamento das doenças respiratórias inflamatórias, desde que seja utilizada uma dose suficientemente eficaz. Em conclusão, foram realizados dois estudos laboratoriais e quatro estudos epidemiológicos focados nos efeitos da adenosina em doença respiratória humana e obteve-se evidência que suporta um papel relevante das purinas na patogénese das doenças pulmonares inflamatórias humanas. Estes resultados sustentam a realização de ensaios clínicos com agonistas dos recetores A2A da adenosina em doenças pulmonares inflamatórias.
Chronic inflammatory lung diseases, such as asthma, chronic obstructive pulmonary disease and interstitial lung diseases are a major and rising global source of disability and early death. An urgent need for new therapies is widely recognized, but the identification of therapeutic targets in in vitro and animal studies has not been successfully translated into effective human treatments. The modulation of purinergic receptors is a particularly promising new strategy for the control of inflammation and promotion of homeostasis. A breadth of preclinical data has shown that adenosine receptors are active in cells and tissues with a role in pulmonary inflammation and their modulation leads to functional improvements in animal models. The application of these strategies in clinical trials was, however, disappointing. Some authors argue that this lack of effectiveness is related to inter-specific differences in distribution and effects of adenosine receptors. This study aimed to assess the effects of adenosine receptors in human disease. The final objective was to obtain a proof-of-concept for the use of adenosine receptor modulation for the treatment of human disease. A three-step approach was used. First the effects of chronic consumption of caffeine, a non-selective antagonist of the A1 and A2A adenosine receptors, were studied, allowing for a non-interventional evaluation of their effects on human respiratory disease. The levels of an adenosine precursor in the lung were then assessed, allowing for an appraisal of the expected endogenous activation of purinergic receptors in human disease. Finally, a laboratory method was developed for the study of a robust marker of cell activation in fresh, unchanged human alveolar macrophages, which have a prominent role in the pathogenesis of inflammatory pulmonary disease. The epidemiological studies showed that caffeine intake has positive effects in some lung diseases, such as asthma and post-infectious cough, as well as in lung function and respiratory mortality. The results on COPD and sarcoidosis were less clear. No correlation was found between caffeine consumption and the risk or severity of sleep apnoea. The measurement of the lung levels of adenosine triphosphate (ATP), revealed higher levels in patients with sarcoidosis compared to those with hypersensitivity pneumonitis, suggesting that ATP could useful as a diagnosis biomarker. Finally, adenosine A2A receptors underwent adaptive changes of density and their activation reduced the intracellular free calcium transients resulting from exposure to an inflammatory stimulus in human alveolar macrophages. This supports the contention that adenosine A2A receptors are a promising therapeutic target for treating inflammatory lung disorders in humans, provided that a sufficiently effective dose of agonist is used. In conclusion, these two laboratory and four epidemiological studies focused on the effects of adenosine in human lung disease found evidence that supports a relevant role for purines in the pathogenesis of human inflammatory lung disease. These results support the further testing of adenosine A2A receptor agonists for the treatment of inflammatory lung diseases.
This project was funded by Fundação para a Ciência e Tecnologia (FCT) through Grants SFRH/BD/69640/2010 (funded by POPH-QREN) and project grant PIC/IC/83290/2007, which is supported by FEDER (POFC e COMPETE) (PIC/IC/82390/2007) and FCT.

Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Major depressive disorder (MDD), the most prevalent mental illness, is a chronic and recurrent condition (Lucas et al., 2011). MDD seems to be associated with abnormalities in regions that mediate emotional and stress responses (Manji et al., 2001). A strong link between suicide and depression has been showed, with more than 86% of suicide victims having a depressive disorder, and committing suicide most often after a major depressive episode (Coryell & Young, 2005; Rihmer, 2007). So, it is possible to study the brain tissue from suicide completers, in order to understand the neurobiological basis of depression. Suicide has been identified as a serious public health problem. Risk factors, such as biological, psychiatric and cultural, interact in a complex manner to this pathology (Bertolote & Fleischmann, 2005). Adenosine is an endogenous nucleoside that influences many functions in the central nervous system (CNS) (Fredholm et al., 2005), acting as a homeostatic modulator and also as a neuromodulator at the synaptic level, where it modulates the release of neurotransmitters, the post-synaptic responsiveness and the action of other receptor systems (Cunha, 2001). Adenosine acts via activation of four G-protein coupled receptors (GPCRs) (Fredholm, 1997), in the brain, it acts especially through activation of two adenosine receptors (ARs), inhibitory A1 (A1R) and facilitatory A2A (A2AR) receptors (Cunha, 2005; Wei et al., 2011). The interest in the role of adenosine in mood disorders arised from previous studies that have recognized a relationship between caffeine intake, mood changes and specific psychiatric symptoms (Kawachi et al., 1996; Lara, 2010; Lucas et al., 2011; Lucas et al., 2013), since caffeine has biological effects as competitive antagonist of A1R and A2AR (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2003; Fredholm et al., 2005; Fredholm, 2007). There is also evidence that different therapeutic strategies used to control mood disorders are related to the adenosine modulation system (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2001; Gomes et al., 2011). In animal models of manipulation of ARs, there are modified behavioral responses considered relevant for mood in humans (Gomes et al., 2011). The main goal of this study was to understand the differences in the localization, density and function of both A1R and A2AR between control subjects and suicide completers, in brain areas affected by depression. Also, we aimed to comprehend if these alterations are related with synaptic changes. For this purpose, a postmortem study was performed in male subjects who died by suicide, aged-matched with nonsuicide controls who died by natural causes or accidents. Human brain samples were obtained at autopsies performed in Instituto Nacional de Medicina Legal e Ciências Forenses, I. P. (INMLCF), Coimbra, Portugal. Several brain areas were studied: Brodmann area 25 (BA25); medial caudate nucleus (MC); posterior caudate nucleus (PC) and hippocampus. To comprehend the normal localization of A1R and A2AR we compared the relative abundance of A1R and A2AR in total extracts (TE) and in nerve terminals (NT), isolated using an adapted discontinuous Percoll gradient protocol (Dunkley et al., 1986, 2008), from human brain areas of the same sample. In all brain areas studied, both receptors were found enriched in NT. We then refined the information on ARs subsynaptic localization, using a fractionation method (Phillips et al., 2001; Rebola et al., 2005), and it was observed that A2AR are mainly located outside the active zone and A1R are present in all synaptic fractions. Synaptic changes present in the brain of suicide completers were then studied and it was observed a down-regulation of synaptosomal-associated protein 25 (SNAP25) on BA25 and MC together with a decrease in postsynaptic density protein 95 (PSD95) levels on PC and hippocampus. Astrocytic marker glial fibrillary acidic protein (GFAP) down-regulation was observed in BA25 and hippocampus. Several alterations in the density of ARs, in the different brain regions, were observed in suicide completers when compared with age-matched controls. We described: an up-regulation of A2AR in TE, on BA25 and PC; an up-regulation of A1R in TE, on MC; a down-regulation of A1R in NT, on PC. Due to their particular distribution in the brain and their functional properties, ARs constitute an attractive opportunity for developing innovative compounds for the treatment of specific neurodegenerative and psychiatric disorders, such as MDD. Our work has provided information about changes present in the brains of suicide completers and might contribute to better understand the modulatory role of ARs in depression. There are still numerous questions that demand careful attention to further explore this system and develop novel strategies to control mood disorders, particularly MDD.
A perturbação depressiva major (MDD), a mais predominante das doenças mentais, é uma condição crónica e recorrente (Lucas et al., 2011). A MDD parece estar associada com alterações nas regiões que medeiam as respostas emocionais e de stress (Manji et al., 2001). Uma forte ligação entre o suicídio e a depressão foi demonstrada, uma vez que mais de 86% das vítimas de suicídio apresentavam uma perturbação depressiva, na maioria dos casos, ocorrendo o suicídio depois de um episódio depressivo major (Coryell et al., 2005; Rihmer, 2007). Assim, o tecido cerebral de vítimas de suicídio pode ser utilizado para estudar a neurobiologia da depressão. O suicídio foi identificado como um problema sério de saúde pública. Vários fatores de risco biológicos, psiquiátricos e culturais interagem de uma maneira complexa para esta patologia (Bertolote & Fleischmann, 2005). A adenosina é um nucleósido endógeno que influencia muitas funções do sistema nervoso central (CNS), agindo como modulador homeostático e também como neuromodulador ao nível sináptico, onde modula a libertação de neurotransmissores, a capacidade de resposta pós-sináptica e a ação de outros sistemas receptores (CNS) (Cunha, 2001; Fredholm et al., 2005). A adenosina actua em quatro receptores acoplados à proteína G (GPCRs) (Fredholm, 2007), no cérebro age especialmente através da activação de dois receptores de adenosina (ARs), inibitório A1 (A1R) e facilitatório A2A (A2AR) receptores (Cunha, 2005; Wei et al., 2011). O interesse do papel da adenosina nos distúrbios de humor surgiu de numerosos estudos que reconheceram uma relação entre o consumo de cafeína, alterações de humor e sintomas psiquiátricos específicos (Kawachi et al., 1996; Lara, 2010; Lucas et al., 2011; Lucas et al., 2013), uma vez que a cafeína tem efeitos biológicos como antagonista competitivo dos A1R e A2AR (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2003; Fredholm et al., 2005; Fredholm, 2007). Também há evidências de que as estratégias terapêuticas utilizadas para controlar distúrbios de humor estão relacionados com o sistema modulatório de adenosina (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2001; Gomes et al., 2011). Em modelos animais de manipulação de ARs há respostas comportamentais consideradas relevantes para o humor nos humanos (Gomes et al., 2011). O principal objectivo deste estudo era perceber as diferenças na localização, densidade e função dos A1R e A2AR, entre vítimas de suicídio e controlos, em áreas afectadas pela depressão. Além disso, tentámos compreender se essas alterações estão relacionadas com modificações sinápticas. Para este propósito foi realizado um estudo postmortem em sujeitos do sexo masculino suicidas, pareados com controlos da mesma idade que morreram de causas naturais ou acidentes. As amostras de cérebro humano foram obtidas em autópsias realizadas no Instituto Nacional de Medicina Legal e Ciências Forenses, I. P. (INMLCF), Coimbra, Portugal. Foram estudadas diversas áreas cerebrais: área de Brodmann 25 (BA25); núcleo caudado médio (MC); núcleo caudado posterior (PC) e hipocampo. Para compreender a localização normal dos A1R e do A2AR comparámos a abundância relativa dos ARs em extratos totais (TE) e em terminais nervosos (NT), isolados usando um protocolo adaptado de gradiente de Percoll discontínuo (Dunkley et al., 1986, 2008), das áreas cerebrais da mesma amostra. Em todas as áreas estudadas os dois receptores encontram-se enriquecidos nos NT. De seguida, refinámos a informação acerca da localização subsináptica dos ARs, usando um método de fracciomento (Phillips et al., 2001; Rebola et al., 2005) e foi observado que os A2AR estão maioritariamente localizados fora da zona activa da sinapse e os A1R estão presentes em todas as fracções sinápticas. Foram então estudadas alterações sinápticas presentes no cérebro de suicidas e foi observada uma diminuição de densidade da synaptosomal-associated protein 25 (SNAP-25) na BA25 e no MC juntamente com um decréscimo nos níveis de postsynaptic density protein 95 (PSD-95) no PC e no hipocampo. Foi observado uma diminuição da densidade do marcador astrocítico glial fibrillary acidic protein (GFAP) na BA25 e no hipocampo. Várias alterações na densidade dos ARs, nas diferentes regiões do cérebro, foram observadas nas amostras de suicidas quando comparadas com os controlos. Nós descrevemos: aumento da densidade de A2AR em TE, na BA25 e no PC; aumento de densidade de A1R em TE, no MC; diminuição de densidade de A1R em NT, no PC. Devido à sua particular distribuição no cérebro e as suas propriedades funcionais, os ARs constituem uma oportunidade atrativa para desenvolver compostos inovadores para o tratamento de distúrbios psiquiátricos e neurodegenerativos, como a MDD. O nosso trabalho forneceu informação sobre as mudanças presentes no cérebro de suicidas e pode contribuir para uma melhor compreensão do papel modulador dos ARs na depressão. Há ainda numerosas questões, que requerem atenção cuidadosa, para continuar a explorar este sistema e desenvolver novas estratégias para controlar os distúrbios de humor, particularmente, a MDD.