Dissertations / Theses on the topic 'P2X3 antagonist'

Purinerge P2X3-Rezeptoren spielen eine bedeutende Rolle in der Vermittlung chronischer Schmerzen, welche ein führendes Problem des Gesundheitswesens mit vielen sozioökonomischen Konsequenzen darstellen. Die Tatsache, dass P2X3-Rezeptoren fast ausschließlich von nozizeptiven Neuronen exprimiert werden, macht sie trotz ihres besonderen Desensitisierungsverhaltens zu vielversprechenden Angriffspunkten zukünftiger Schmerztherapien, beispielsweise mithilfe kompetitiver Antagonisten an diesen Rezeptoren. Zur Analyse der Wechselwirkungen zwischen Agonist und kompetitivem Antagonist wird meist der Schild-Plot benutzt. Jedoch ist dieser im Falle der sehr schnell desensitisierenden P2X3-Rezeptoren ungeeignet, da die Vorbedingung eines stabilen Gleichgewichts zwischen Agonist und Antagonist aufgrund der Desensitisierung nicht erfüllt ist. Ziel der vorliegenden Arbeit war es, eine neue Methode zur Analyse der Interaktion kompetitiver Antagonisten mit ihrer Bindungsstelle am Beispiel des P2X3-Rezeptors zu entwickeln und so für die Antagonistenbindung bedeutende Aminosäuren der Bindungsstelle zu identifizieren. Mittels der Patch-Clamp-Technik wurden die Effekte der Antagonisten A-317491, TNP-ATP und PPADS auf die vom P2X1,3-Rezeptor-selektiven Agonisten α,β-MeATP induzierten Ströme am P2X3-Wildtyp-Rezeptor und an fünf Rezeptormutanten mit veränderter Ligandenbindungsstelle untersucht. Alle Rezeptoren wurden in HEK293-Zellen exprimiert. Anhand der gemessenen Daten wurde ein Hidden Markov Model (HMM) erstellt, welches die sequentiellen Übergänge des Rezeptors von geschlossen zu offen und desensitisiert in An- und Abwesenheit des Antagonisten miteinander kombiniert. Die am P2X3-Rezeptor induzierten Ströme konnten mithilfe dieses Modells korrekt gefittet und die für die Antagonistenbindung wichtigen Aminosäuren innerhalb der Bindungsstelle bestimmt werden. Als Resultat dieser Arbeit konnte außerdem gezeigt werden, dass das HMM eine geeignete Methode zur Analyse der Wirkung kompetitiver Antagonisten an schnell desensitisierenden Rezeptoren darstellt. Die untersuchten Antagonisten A-317491 und TNP-ATP haben einen kompetitiven Wirkmechanismus, während PPADS eine pseudoirreversible Blockade verursacht.

Structural information for the zebrafish P2X4 receptor in both an agonist bound and unbound resting state provided a major advance in understanding agonist action and has given insight into movement that occurs in the receptor upon ATP binding. Despite agonist action now being well characterised, the molecular basis of antagonism is poorly understood. In this thesis the mechanism of antagonist action at the hP2X1 receptor has been investigated through determining properties of chimeras and mutant receptors based on differences between antagonist sensitive and insensitive P2X receptors. The antagonists suramin, NF449 and PPADS potently inhibit the human P2X1 receptor but have little or no action at the rat P2X4 receptor. The extracellular loop of the hP2X1 receptor was shown to determine antagonist sensitivity and was therefore split into four sections, residues of which were swapped with corresponding residues of the antagonist insensitive rP2X4 receptor and vice versa. Sub-chimeras and point mutations were then made to identify particular residues and regions which contribute to antagonist action. These experiments identified two regions important for NF449 binding at the receptor. These are a cluster of four positively charged residues at the base of the cysteine rich head region (136-140) and three residues located just below them (T216, H224 and Q231). An NF449 bound model of the hP2X1 receptor has been generated. The introduction of the four positively charged residues at the base of the cysteine rich head region to the rP2X4 receptor introduced suramin and PPADS sensitivity to this previously insensitive receptor. This mutation is thought to cause a conformational change which allows the antagonist to bind at residues which are already present in the wildtype receptor. In summary this thesis has advanced the understanding of antagonist action at the hP2X1 receptor and the antagonist insensitivity of the rP2X4 receptor.

ABSTRACT BACKGROUND AND AIMS: The P2X7 receptor (P2X7R) is an ATP-gated, non-selective cation channel highly expressed on monocytes, macrophages and microglia. Prolonged activation of the P2X7R by ATP leads to cytolytic pore formation and the release of inflammatory mediators including interleukin-1β and prostaglandin E2. Accumulating evidence suggests a role for the P2X7R in neuroinflammation and thus P2X7R antagonists might be useful in diseases including chronic pain, depression and Alzheimer’s disease. Both negative allosteric modulators of the P2X7R, such as the adamantyl benzamides, and orthosteric antagonists, such as the aryl cyanoguanidines, inhibit the ATP-induced release of IL-1β from immune cells. This shared ability to inhibit IL-1β release may explain why no attempts have been made to determine the features which promote binding to the allosteric or orthosteric site. An advantage of targeting the allosteric or orthosteric site might emerge however, if a different agonist of the P2X7R is used. An antimicrobial peptide produced within the human body, LL-37, is also able to activate the P2X7R and yet LL-37 is never used in the characterisation of new series of P2X7R antagonists. The aims of this project were to characterise a novel series of P2X7R antagonists, the adamantyl cyanoguanidines, which have a hybrid structure derived from the adamantyl benzamides and aryl cyanoguanidines. Characterisation of the adamantyl cyanoguanidines should allow determination of the features which promote binding to the orthosteric or allosteric site of the P2X7R, which was one of the primary aims of this project. A second aim was to evaluate the potential of the hybrid series for further development as P2X7R antagonists by considering their potency and physicochemical P a g e | 13 properties. The final aim was to determine if there was any advantage of targeting the orthosteric or allosteric site of the P2X7R particularly with regard to inhibiting LL-37-mediated activation of the P2X7R. METHODS: The potency of adamantyl cyanoguanidines and reference P2X7R antagonists were determined in YO-PRO-1 dye uptake assays and interleukin-1β release assays to develop structure-activity relationships. A potent member of the adamantyl cyanoguanidines and several reference P2X7R antagonists were pharmacologically characterised in Schild assays, washout studies and receptor protection studies. The ability of several negative allosteric modulators and an orthosteric antagonist to inhibit LL-37-induced dye uptake was also examined. RESULTS: More compact adamantyl cyanoguanidines including those with a methylene linker between the adamantyl and cyanoguanidine groups and no linker between the cyanoguanidine group and phenyl ring were more potent than analogues with longer linkers. Ortho-substitution of the phenyl ring or substitution of the ring with 5-quinoline led to increased potency. The potency seen in the dye uptake assay was also seen in the interleukin-1β release assay. A potent member of the series 3-19 was determined to be a slowly reversible negative allosteric modulator as was 1-17 an adamantyl benzamide. The parent aryl cyanoguanidine, A-804598, was confirmed to be an orthosteric antagonist. None of the compounds were able to inhibit LL-37-induced dye uptake. DISCUSSION AND CONCLUSIONS: The determined structure-activity relationships for the adamantyl cyanoguanidines confirm their potential for further development since the series was highly amenable to modification and several potent analogues have favourable physicochemical properties including lower molecular weight. Since 3-19 P a g e | 14 was a negative allosteric modulator despite its structural similarity to A-804598 this suggests the adamantyl group promotes binding to the allosteric site and that cyanoguanidine is a tolerated bioisostere for the acetamide group at the allosteric site. The 5-quinolinyl group, in the appropriate position, facilitates binding to either site. The failure of multiple P2X7R antagonists to inhibit LL-37-induced dye uptake is concerning since LL-37 alone has been shown to induce the release of interleukin-1β from human monocytes. Future research must determine if LL-37 is responsible for cytokine release in vivo and develop small molecule antagonists of the action of LL-37.

P2X receptors are ATP-gated cation channels. P2X1 receptors are widely expressed throughout the body and have a range of functional roles, e.g. contraction of mesenteric arteries and regulation of blood clotting. The recent crystallisation of the zebra fish P2X4 receptor has provided a major advance in understanding the molecular basis of receptor properties. However, how agonists or antagonists are co-ordinated and the extent of the proposed ligand binding site have not been addressed at a structural level. A mutagenesis based approach was used to propose a model of the ATP binding site and has highlighted some residues involved in antagonist action at P2X receptors. The aim of this thesis was to investigate the molecular basis of antagonist action at the P2X1 receptor using site-directed mutagenesis and P2X receptor chimeras. The wild-type, mutant and chimaeric P2X receptors were expressed in Xenopus laevis oocytes and the currents were characterised using two electrode voltage clamp. Initially, suramin was shown to act as a competitive antagonist and PPADS as a non-competitive antagonist at the P2X1 receptor. The contribution of residues V74 to G96 to human P2X1 receptor properties were determined using cysteine scanning mutagenesis. This region contains a residue that has been shown to be important in suramin action at the P2X4 receptor (K78) but cysteine mutation of the residues V74 to G96 had either no effect or slightly increased antagonism by suramin or PPADS. Also, a further residue was found to be important in ATP potency (F92) and the use of partial agonists and modification with cysteine reactive methanethiosulfonate (MTS) reagents identified additional residues important in channel activation. Mapping these residues onto a homology model of the P2X1 receptor showed the depth of the agonist binding site and highlighted the importance of the rear/inner cavity of the binding pocket in the gating of the channel subsequent to agonist binding. The cysteine rich head region of the P2X receptor, which is adjacent to the proposed ATP binding pocket, is absent in the antagonist insensitive Dictyostelium receptors. P2X1 and P2X2 receptors have ~1400-fold difference in sensitivity to a suramin analogue NF449. Chimeras and point mutations in the cysteine rich head region were made between the P2X1 and P2X2 receptors and they identified the region between the third and fourth conserved cysteine residues of the P2X1 receptor as being important in conferring the difference in sensitivity. In particular, the positively charged residues at the base of the cysteine rich head region of the P2X1 receptor accounted for the highly selective antagonism of NF449 at the P2X1 receptor. Additionally, these residues were shown to play a role in the molecular basis of suramin and PPADS action at the P2X1 receptor. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity. In silico docking models highlighted possible sites of action for NF449 and suramin on the P2X1 receptor showing that the base of the cysteine rich head region may be important in the binding of antagonists. In summary, this research furthered understanding of ligand action at the P2X1 receptor.

The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders.

P2X receptors (P2XRs) activated by ATP are widely expressed throughout the human body and mediate various physiological and pathophysiological roles. Crystal structures have provided a major advance in understanding agonist and subtype selective antagonist actions. However, the molecular basis of antagonism of general antagonists is poorly understood. PPADS is an effective antagonist at most mammalian P2XRs. Previous studies suggested lysine residue 249 (K249) (numbering for P2X1R) was involved in PPADS action. The aim of this study was to determine the PPADS binding site in a molecular model of the human P2X1R based on the zebra fish P2X4R (zfP2X4R) crystal structure. Contributions of individual residues in a ring centred on K249 with a radius of the length of PPADS were investigated by cysteine mutagenesis. The effect of their cysteine substitutions on accessibility following PPADS binding and on PPADS sensitivity were tested. A cluster of positively charged residues (K70, K190 and K249) at the orthosteric pocket showed decreases in both accessibility and sensitivity to PPADS, suggesting they are directly involved in binding of the antagonist. These data allow validation of molecular docking to provide the first model of PPADS binding. Some residues outside the orthosteric area showed decreased accessibility following PPADS binding but on change in antagonist sensitivity, indicating PPADS binding induced significant conformational changes from the apo state. In addition, the charge and conformational changes at the cysteine rich head (CRH) region also contributed to antagonist action by showing a mutation at a positively charged residue (K138C) increased accessibility following PPADS binding and decreased PPADS sensitivity. In summary, this thesis has advanced the understanding of antagonist PPADS action and provided a template to develop subtype selectivity based on the differences between subunits around the orthosteric P2XR binding site and the CRH region.

P2X4 is a purinergic receptor distributed all over the body with various roles. Among them, it was reportedly overexpressed in several neuronal and immune cell types following peripheral nerve injury and its activation leads to neuropathic pain. Several compounds were found to block P2X4 but none has gone into a clinical stage, probably due to insufficient information about the compound itself and P2X4 in general. One of such compounds, 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) is known to be widely used in P2X4-related studies despite its limited information. Therefore, this study aimed to (i) find a novel compound that can block the activation of P2X4 through high throughput screening and characterise it, and (ii) study structural-activity relationship between 5-BDBD and P2X4. Human P2X4 receptor was stably expressed in human 1321N1 astrocytoma cells and 1710 compounds from National Cancer Institute were screened for their activity at P2X4. Extensive tests led to identifying a natural product (thaspine) as the most potent inhibitor at reducing P2X4 activation. Further characterisation experiments revealed that thaspine had an IC50 value of 3.8 ± 0.2 μM and showed an allosteric mode, time-dependent and irreversible inhibition. Thaspine was similarly potent at mouse P2X4, but not effective at human P2X2, P2X2/3 and P2X7. It was also inactive at human P2Y2 and P2Y6 at concentrations below 10 μM and 30 μM respectively. In primary microglial cell model (BV2), it inhibited ivermectin-potentiated responses but not normal ATP-evoked responses. Meanwhile, 5-BDBD was found to be inhibiting P2X4 receptor competitively and diazepinone was a pivotal group of the structure to cause inhibition. The binding pocket of 5-BDBD at P2X4 was also predicted using molecular docking. In this study, a novel compound thaspine, has been shown to be effective at inhibiting human P2X4 and thus may have potential therapeutic applications while novel information about 5-BDBD was also acquired.

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O P2X7 é um receptor purinérgico que está envolvido em importantes funções fisiológicas e metabólicas, mas também tem participação em diversas patologias, principalmente aquelas de caráter inflamatório. Apesar de sua relevância, ainda não se têm disponíveis antagonistas específicos que possam ser utilizados na clínica para o tratamento de doenças relacionadas à ativação deste receptor. Muitos fármacos comercializados nos dias de hoje apresentam estruturas químicas relacionadas a um produto natural extraído de alguma espécie botânica de uso consagrado na medicina popular. Baseia-se nisso a relevância do estudo de produtos naturais para obtenção de uma atividade específica sobre alvos celulares. Logo, esse trabalho teve como interesse o estudo de extratos vegetais e fúngicos obtidos de espécies dos diferentes biomas, cedidos pelo LQPN do Centro de Pesquisas René Rachou \2013 Fiocruz-MG, visando á identificação de antagonistas para o receptor P2X7. Nosso primeiro passo foi à padronização de uma metodologia que permitiu a triagem de cerca de 60 extratos ao mesmo tempo, através da utilização de um espectrofotômetro de placas. Depois de padronizada, promovemos a aplicação dessa metodologia na triagem de 1800 extratos, dos quais apenas três extratos (8067,8549 e 8568) apresentaram atividade antagonista na faixa de corte pré-estabelecida [100 \03BCg/mL], com perfis de inibição de 65 %, 62 % e 61 % respectivamente, sobre o P2X7R. Destes extratos foram determinados os IC´s50 tanto em células de linhagem murina (2,1 \03BCg/mL; 2.6 \03BCg/mL e 3.8 \03BCg/mL) quanto em células de linhagem humana (0.69, 0.92 e 1.5 \03BCg/mL), sendo possível verificar maior atividade quando testados em células de linhagem humana Posteriormente avaliamos a ação destes sobre funções fisiológicas relacionadas à ativação do P2X7R, nessa etapa, observamos o efeito inibitório destes extratos sobre a liberação de IL-1beta, ROS e NO, onde os três compostos foram capazes de inibir estas funções numa faixa entre 50 % a 60 %. Para obtermos uma caracterização do efeito farmacológico destes extratos sobre o receptor P2X7, realizamos experimentos de eletrofisiologia, caracterizando assim uma ação inibitória dose-dependente destes, sendo que nos respectivos IC´s50 os perfis de inibição da corrente foram de: 76 %, 47 % e 75 %. Também avaliamos a citotoxicidade in vitro utilizando as células de ambas às linhagens e verificamos que não apresentaram significativa toxicidade quando tratadas por 24 h. em doses até quatro vezes maiores que o IC50, visto que os resultados foram semelhantes ao controle não tratado. Depois de avaliarmos a atividade antagonista destes extratos in vitro, partimos para os experimentos in vivo utilizando os modelos de úlcera induzida por etanol e de dor neuropática e inflamatória, pois existem trabalhos previamente descritos na literatura que correlacionam a atividade do P2X7R com a evolução dessas patologias. No ensaio de dor neuropática, apenas dois extratos mostraram atividade analgésica (8067 e 8549) inibindo o estímulo de dor em 68 % e 66 %, porém no contexto da dor inflamatória os três extratos mostraram efeito analgésico, inibindo o estímulo em: 8067 = 48%, 8549 = 50% e 8568 = 44 % Os resultados obtidos do experimento de úlcera induzida por etanol demonstraram o efeito inibitório sobre a formação de úlceras desses extratos em: 88 %, 84 % e 51 %, inclusive foram mais efetivos que o BBG (antagonista reversível deste receptor) e que o medicamento utilizado na clínica (Lansoprazol), os quais inibiram a formação de úlceras em 43 % e 46% respectivamente. Nosso conjunto de resultados apontam extratos com significativa atividade antagonista sobre o P2X7R, com potencial para o desenvolvimento de novos fármacos com grande interesse para a indústria farmacêutica, além de contribuir para o conhecimento acerca de propriedades medicinais presentes na biodiversidade
The purinergic receptor P2X7 is involved in important physiological and metabolic functions, but it also participates in pathology, especially when inflammatory in character. Despite the importance of P2X7, it has no specifi c antagonists yet available for use in clinical treatment of diseases related to the receptor's activation. Today, many drugs on the market have chemical structures related to natural products obtained from botanical species with traditional use in indigen ous medicine, forming the basis for studying natural products to obtain specific activity on cellular targets. This work focused primarily on the study of plant and fungal species extracts obtained from different biomes provided by LQPN of the Research Cen ter René Rachou - Fiocruz - MG, aiming to identify antagonists for the P2X7 receptor. The first step was to standardize a method that allowed the screening of approximately 60 extracts at the same time through the use of a plate spectrophotometer. Once stand ardized, the application of this methodology was promoted in the screening of 1800 extracts. Of these, only three extracts (8067, 8549 and 8568) showed antagonistic activity in the pre - established cut range [100 mg/mL], with inhibition profiles of 65%, 62% , and 61% respectively, on the P2X7R. Th e IC's50 of them were determinate in murine (2.1, 2.6, and 3.8 mg/mL) and human (0.69, 0.92, and 1.5 mg/mL) cell lines. Which an increased activity was possible to verify when they were tested in human cells. Consequ ent evaluation of action on physiological functions related to the activation of P2X7R revealed an inhibitory effect of these extracts on the release of IL - 1beta, NO, and ROS. The three tested compounds were able to inhibit these functions in a range betwe en 50% and 60%. To obtain a pharmacological characterization of these extracts on the P2X7 receptor, electrophysiological experiments were conducted, which characterized the dose - dependent inhibitory effects, exhibiting inhibitory current profiles of 76%, 47%, and 75%, respectively. In vitro cytotoxicity was also evaluated, using both cell strains, showing no significant toxicity after 24 hours of treatment at doses of up to four times that of the IC50; the results were similar to the untreated control. Aft er evaluating the antagonistic activity of these extracts in vitro, experiments using the in vivo models of ethanol - induced ulcers and inflammatory and neuropathic pain were performed. Previous studies correlate the activity of the P2X7R with the evolution of these pathologies. In the neuropathic pain protocol experiment, only two extracts showed analgesic activity (8067 and 8549) by inhibiting the pain stimulation by 68% and 66%, but in the context of inflammatory pain, the three tested extracts showed ana lgesic effects by inhibiting the stimulus by the following percentages: 8067 = 48 %, 8549 = 50%, and 8568 = 44%. The results of the ethanol - induced ulcer demonstrated an inhibitory effect on the ulcer’s development of 88%, 84%, and 51% by these extracts, a nd found that the extracts were more effective than BBG (reversible antagonist of this receptor) and the medicine used clinically (Lansoprazole), which inhibited the formation of ulcers by 43% and 46%, respect ively. The data set links to extracts with significant antagonist activity on the P2X7R, and potential to the development xix of new medicines of great interest to the pharmaceutical industry and important contributions to the knowledge of medicinal properties present in biodiversity

O objetivo deste estudo foi determinar se a variação genética no gene do receptor P2X7 está associada com a diminuição da densidade mineral óssea e o risco de osteoporose em pacientes acima de 50 anos de idade com fratura de tornozelo. Foi realizado um estudo diagnóstico Nível I. Os pacientes acima de 50 anos com fratura de tornozelo submetidos ao tratamento cirúrgico foram divididos em dois grupos após o resultado da densitometria óssea: o grupo de estudo com osteopenia (T score entre -1 e -2,5) ou osteoporose (T score <= -2,5) e o grupo controle com valores de normalidade (com T score >= -1). Os critérios de exclusão foram alterações que levam à osteoporose secundária. Os pacientes foram genotipados para 15 polimorfismos de nucleotídeo único (SNPs) não sinônimos dentro do receptor P2X7 (numerados de 1 à 15) obtidos a partir da saliva. Avaliamos 121 pacientes com fratura de tornozelo, sendo 56 do grupo controle e 65 do grupo de estudo. Todos os pacientes eram sedentários, não utilizavam nenhum medicamento para tratamento de osteoporose, não eram tabagistas e sofreram trauma de baixa energia. A análise agrupada das alterações dos SNPs demonstrou que se o gene tem 3 ou mais variantes de SNPs (36,4% dos 121 pacientes), dos 15 possíveis, ele está alterado com repercussão clínica relacionada à perda ou ganho de função do gene. E ao analisar as alterações dos SNPs, individualmente, os resultados sugerem que: os SNPs 1,4,14 e 15 são variantes de perda de função; SNPs 5 e 10 são descritos como variantes de perda de função; entretanto, não têm influência na nossa população; SNPs 11 e 13 são variantes de perda de função e não ganho de função, como descrito na literatura; e SNP 12 foi associado à perda de função em nossa população. Podemos ressaltar como limitações do nosso estudo o fato de nos concentramos principalmente em polimorfismos não sinônimos que não cobrem toda a variação genética em P2X7 e no número pequeno de participantes quando comparados com a literatura mundial. Em contrapartida, um dos pontos fortes do nosso estudo é ser o primeiro a avaliar o P2X7 na população brasileira, que é bastante heterogênea do ponto de vista genético devido à nossa miscigenação, quando comparado com os outros estudos que avaliaram a população do norte da Europa, que é mais homogênea geneticamente. Em conclusão, o polimorfismo do SNP 12 em P2X7 está associado à densidade mineral óssea e risco de fraturas de tornozelo
The purpose of this study was to determine whether a genetic variation in the P2X7 receptor gene is associated with reduced bone mineral density and the risk of osteoporosis in patients over 50 years of age with ankle fractures. A Level-1 diagnostic study was conducted. Patients over 50 years of age with ankle fractures who had undergone surgical treatment were divided into two groups following the result of a bone densitometry: a study group with osteopenia (bone mineral density T score between -1 and -2.5) or osteoporosis (bone mineral density T score <= -2.5) and the control group with normal values (bone mineral density T score >= -1). Exclusion criteria were alterations that led to secondary osteoporosis. Patients were genotyped for 15 nonsynonymous single nucleotide polymorphisms (SNPs) within the P2X7 receptor (numbered from 1 to 15) obtained from saliva. We evaluated 121 patients with ankle fractures, 56 being from the control group, and 65 from the study group. All patients were sedentary, did not take any medication for the treatment of osteoporosis, did not smoke, and had suffered a low-impact trauma. The grouped assessment of the SNP alterations showed that if a gene has three or more SNP variants (36.4% of the 121 patients), out of the 15 possibilities, it is altered with clinical repercussions related to the loss or gain of the function of the gene. In evaluating the SNP alterations individually, the results suggest that: SNPs 1,4,14, and 15 are loss of function variants; SNPs 5 and 10 are described as loss of function variants; however, they have no influence on our study population; SNPs 11 and 13 are loss of function variants and not gain of function function as is described in the literature; and SNP 12 was associated with a loss of function in our population. In conclusion, we showed that the functional polymorphisms in P2X7 are associated with Bone Mineral Density and the risk of ankle fractures. As limitations to our study, we can point out the fact that we focused mainly on nonsynonymous polymorphisms, which do not cover all the genetic variations in P2X7, and the small number of participants when compared to the world literature. On the other hand, a strength of our study is that it was the first to assess P2X7 in the Brazilian population, which is quite heterogeneous from the genetic point of view due to our miscegenation, as compared to other studies that evaluated the population of northern Europe, which is genetically more homogeneous. In conclusion, the SNP12 polymorphism in P2X7 is associated with Bone Mineral Density and the risk of ankle fractures

CARMO, Marta Regina Santos do. Efeito neuroprotetor do antagonismo do receptor P2X7 no Parkinsonismo experimental induzido por 6-OHDA. 2015. 110 f. Tese (Doutorado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015.
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Parkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and a concomitant decrease of dopamine (DA) in the striatum, which can be modeled by 6-OHDA administration. Since ATP released from damaged cells can exert noxious effects on neurons, acting through its P2X7 receptors (P2X7R), the aim of the present study was to investigate the effects of a P2X7R antagonist, Brilliant Blue G (BBG) on 6-OHDA-induced neurotoxicity. Male Wistar rats received stereotaxic injections of 6-OHDA (18 µg/3µl) into the right striatum and were treated with BBG (45 mg/kg, i.p. 48/48 h) for two weeks. In an additional experiment, animals were treated with the selective antagonist A-438079 (10 µM i.c.v.) for two weeks. BBG decreased the number of contralateral rotations in the apomorphine test, an effect mimicked by the selective P2X7R antagonist A438079. BBG has also improved the animals’ performance in the passive avoidance test (short-term memory) and in the cued version of the Morris Water maze. The antagonism of P2X7R by BBG has also prevented the reduction of dopamine content in the striatum and SN as well as the loss of dopaminergic neurons, and microgliosis and astrogliosis in the striatum. BBG treatment also decreased the IL-1β levels in striatum, despite the observed effect not being statistically significant. To grasp the mechanism of action of BBG, we used in vitro models exploring synaptotoxicity (striatal synaptosomes) and neurotoxicity (dopamine-differentiated SH-SY5Y cells). Besides showing that P2X7R are present in striatal dopaminergic terminals, we observed that BBG 100 nM prevented the 6-OHDA-induced synaptosomal dysfunction. Furthermore, we have shown the presence of P2X7 receptors in SH-SY5Y cells, their co-localization with tyrosine hydroxilase, and that BBG attenuates the cell damage, evaluated through lactate dehydrogenase release. The present results suggests that P2X7R contribute to PD pathogenesis through a triple impact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7R antagonists in the disease.
A doença de Parkinson (DP) é caracterizada por uma degeneração progressiva dos neurônios da substância negra (SN) e uma concomitante diminuição do conteúdo de dopamina no estriado, que pode ser mimetizada pela administração de 6-OHDA. Visto que o ATP liberado das células danificadas exerce efeitos deletérios diretos sobre os neurônios, agindo através de receptores P2X7, o objetivo do presente trabalho foi estudar os efeitos do Brilliant Blue G (BBG), um antagonista dos receptores P2X7, sobre a neurotoxicidade induzida por 6-OHDA. Ratos Wistar machos receberam injeções estereotáxicas de 6-OHDA (18 µg/3µl) no estriado direito e foram tratados com BBG (45 mg/kg, i.p. 48/48hs) durante 14 dias. Em uma série adicional de experimentos, os animais receberam o antagonista seletivo A-438079 (10 µM i.c.v.) por 14 dias. O tratamento com BBG diminuiu o número de rotações contralaterais no teste da apomorfina, efeito que foi mimetizado pelo antagonista seletivo A-438079. O BBG também melhorou o desempenho dos animais no teste da esquiva passiva (memória de curta duração), assim como na versão com plataforma sinalizada do labirinto aquático. O antagonismo dos receptores P2X7 pelo BBG preveniu ainda a redução do conteúdo de dopamina no estriado e SN, assim como a perda de neurônios dopaminérgicos, a microgliose e astrogliose no estriado. O tratamento com BBG também diminuiu os níveis de IL-1β no estriado, apesar das diferenças observadas entre os grupos não serem estatisticamente diferentes. Com o objetivo de entender melhor o mecanismo de ação do BBG, foram utilizados modelos in vitro explorando a sinaptotoxicidade (sinaptossomas estriatais) e neurotoxicidade (células SH-SY5Y dopaminérgicas diferenciadas). Além da demonstração da presença dos receptores P2X7 nos terminais nervosos estriatais, foi observado que o BBG 100 nM preveniu a disfunção sinaptossomal. Também demonstramos a presença dos receptores P2X7 nas células SH-SY5Y, assim como sua co-localização com tirosina hidroxilase, onde o pré-tratamento com BBG 100 nM diminuiu o dano celular, avaliado através da liberação de lactato desidrogenase. Os resultados obtidos no presente estudo sugerem que os receptores P2X7 contribuem para a patogênese da DP através de um triplo mecanismo sobre a sinaptotoxicidade, gliose e neurotoxicidade, ressaltando o potencial terapêutico dos antagonistas desses receptores na doença.

Salivary gland inflammation is a hallmark of Sjogren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1 beta and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1 beta, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R(-/-)). P2X7R-mediated IL-1 beta release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1 beta release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28(-/-), IFN gamma(-/-), NOD.H-2(h4) mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.

Neste trabalho foram analisados os efeitos do BBG nos neurônios mioentéricos imunorreativos ao receptor P2X7 no íleo de ratos submetidos à isquemia e reperfusão (I/R). A isquemia intestinal foi obtida pela oclusão dos vasos ileais por 45 minutos, com reperfusão de 0 hora (h), 24 h e 14 dias. O BBG foi aplicado nos grupos I/R 24 h e I/R 14 dias nas dosagens de 50 e 100 mg/Kg. O grupo I/R 0 h é o grupo sem reperfusão. Os tecidos foram preparados para análises de duplas marcações, western blotting, histoquímica da mieloperoxidase, histologia e motilidade intestinal. Os resultados demonstraram a presença do receptor P2X7 nos neurônios mioentéricos. Houve uma diminuição da densidade e da área do perfil dos neurônios mioentéricos nos grupos I/R e nos grupos com o BBG houve uma recuperação dos neurônios. Nos grupos I/R houve aumento na expressão do receptor P2X7 e no número de neutrófilos e diminuição da motilidade. Os resultados sugerem que a I/R afetou os neurônios mioentéricos e que o BBG possa ter atenuado os efeitos da I/R, demonstrando uma provável neuroproteção.
We analyzed the effects of BBG antagonist on the P2X7 receptor and rats ileum enteric neurons subjected to I/R. Intestinal ischemia was obtained by ileal vessels obstruction for 45 minutes, followed by reperfusion of 0 h, 24 h and 14 days. The BBG was applied in I/R 24 h and 14 days groups in dosages of 50 and 100 mg/kg. The I/R 0 h is the group without reperfusion. Tissues were prepared by double labeling, western blotting, myeloperoxidase reaction, histology and intestinal motility analyzes. Our results demonstrated the presence of the P2X7 receptor in myenteric neurons. There was a decrease in density and in the area of the cell body profile of the myenteric neurons in the I/R groups and recovery in the BBG groups. In I/R groups there was an increase in the expression of P2X7 receptor and in the number of neutrophils and there was a decrease in intestinal motility and recover in the BBG groups. The results suggest that I/R affect the myenteric neurons and that the BBG may have attenuated the effects of ischemia, thus demonstrating a possible neuroprotection.

Les maladies ischémiques, causées principalement par des thromboses artérielles, sont la première cause de mortalité dans les pays développés. Les antiagrégants plaquettaires sont des médicaments destinés à empêcher la formation des thromboses artérielles. Une stratégie thérapeutique consiste à inhiber les récepteurs responsables de l’activation des plaquettes : le récepteur P2Y12 et le récepteur P2Y1. Pour obtenir une réponse optimale d'agrégation des plaquettes, l'activation synergique des deux récepteurs P2Y1 et P2Y12 a été démontrée. Le travail décrit dans ce mémoire concerne la synthèse de ligands bivalents, c’est à dire le développement d’une seule entité chimique qui combine deux antagonistes ciblant ces deux récepteurs qui pourrait ainsi présenter un intérêt en assurant un effet antiplaquettaire efficace tout en préservant une capacité hémostatique suffisante. Après avoir développé une série d’analogues du cangrelor présentant une chaîne thioalkylée en position C-2 par substitution de 2-halogénonucléosides, les tests d’agrégation plaquettaire ont révélé que ces composés ont un effet inhibiteur sur l’agrégation induite par l’ADP, néanmoins uniquement à forte concentration. La préparation d’un précurseur du MRS2279 qui a pour but de cibler sélectivement le récepteur P2Y1, nous a permis d’envisager la synthèse d’hétérodimères susceptibles d’inhiber les deux récepteurs P2Y12 et P2Y1. En combinant trois « parties » : une adénosine pour la partie OUEST, un bras espaceur (cystamine, di-, tri- et tétraéthylène glycol) et un précurseur du MRS2279 pour la partie EST, nous avons effectué la synthèse de plusieurs ligands bivalents originaux non phosphorylés. Les tests d’agrégation plaquettaire des dimères ainsi obtenus montrent que ceux-ci n’ont pas un effet inhibiteur assez puissant pour pouvoir poursuivre les tests biologiques. Cependant la phosphorylation des 2 pharmacophores semble une solution prometteuse quant à l’activité des ligands bivalents
Ischemic diseases due to arterial thrombosis are the leading cause of death in developed countries. Antiplatelet agents are drugs which prevent the formation of arterial thrombosis. A therapeutic strategy consists in inhibiting the receptors responsible for platelet activation: the P2Y12 and P2Y1 receptor. For an optimal response of platelet aggregation, synergistic activation of both receptors has been demonstrated. The work described in this thesis deals with the synthesis of bivalent ligands, i. E the development of a single chemical entity that combines two antagonists targeting these receptors. This strategy may be conjugating an effective antiplatelet effect along with a sufficient hemostatic capacity. A serie of analogues of cangrelor, which are thioalkylated at C-2 position, were synthesized from 2-halogenonucleosides. The platelet aggregation tests on those compounds revealed an inhibitory effect on aggregation induced by ADP, but only in high concentrations. The preparation of a precursor of MRS2279 (a selective P2Y1 antagonist), allowed us to focus on the synthesis of heterodimers which may inhibit both receptors P2Y1 and P2Y12. Toward this goal, we combined three parts: an adenosine for the Western part, a linker (cystamine, di-, tri-and tetraethylene glycol) and a precursor of MRS2279 for the Eastern part. We performed the synthesis of several original bivalent ligands which are no phosphorylated. The dimers platelet aggregation tests show that they have no sufficient inhibitory effect to continue the biological tests. However, the phosphorylation of the two pharmacophores appears a promising approach for the activity of those bivalent ligands

FONTELES, A. A. O Brilliant blue G, antagonista do receptor P2x7, diminui a discinesia induzida por L-DOPA em animais submetidos a modelo experimental de doença de Parkinson. 2017. 118 f. Tese (Doutorado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017.
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Dyskinesia is a serious motor complication associated with the prolonged administration of L-DOPA in patients with Parkinson’s disease. The peak of L-DOPA-induced dyskinesia (LID) greatly coincides with the maximum action of L-DOPA following its administration. Accumulating evidence supports the fact that ATP controls dopamine release in striatum through P2X7 receptor. The aim of this work was to investigate a high affinity P2X7 receptor antagonist (Brilliant Blue G) in ameliorating L-DOPA-induced dyskinesia. We selected a established 6-OHDA animal model of Parkinson’s disease. One hundred and six animals were divided into 7 groups as following: 1. sham-operated, 2. sham-operated treated with BBG (45 mg/kg i.p.), 3. sham-operated treated with L-DOPA (30 mg/kg), 4. exposed to 6-OHDA only, 5. exposed to 6-OHDA and treated with L-DOPA, 6. exposed to 6-OHDA and treated with L-DOPA and BBG at 22,5 mg/kg, 7. exposed to 6-OHDA and treated with L-DOPA and BBG at 45 mg/kg. Treatments were initiated 15 days following 6-OHDA exposure. L-DOPA treatment was performed daily over a period of 22 days, whereas BBG treatment was performed every other day for the same amount of time. BBG treatment significantly improved AIMs scores at 7, 14 and 21 days following L-DOPA treatment and enhanced the motor coordination performance on rotarod test. Neurodegeneration was assessed through TH-imunohistochemistry and flurojade C staining though BBG did not show neuroregeneration activity and no changes were observed in flurojade C staining. We also evaluated changes in DAT levels, D1 and D2 dopaminergic receptors and DARPP-32 protein and noticed that P2X7 inhibition reestablished DAT levels that were initially reduced in L-DOPA-treated animals, and ameliorated D1 receptor and pDARPP-32Thr34 expression in L-DOPA-treated animals. The neuroinflammation was evaluated through the measurement of GFAP/CD11b/COX-2-imunohistochemistry and IL-1β expression with L-DOPA chronic therapy significantly increasing neuroinflammation in 6-OHDA treated animals, and conversely P2X7 receptor blockade diminishing it. Our results suggest that the purinergic system plays an important role in the pathophysiology of LID, with BBG showing a remarkable anti-dyskinetic effect probably by downregulating D1 pathway activation and controlling neuroinflammation.
A discinesia é o principal efeito adverso do tratamento crônico com L-DOPA em pacientes com Doença de Parkinson. É clinicamente caracterizada por picos de movimentos involuntários anormais coincidentes com o pico de ação da L-DOPA. Visto que o ATP participa no controle da liberação de dopamina no corpo estriado através da ativação de seus receptores, o objetivo deste trabalho foi estudar o possível efeito anti-discinético do antagonista do receptor P2X7, o Brilliant Blue G, na fisiopatologia da discinesia. Foram utilizados 106 ratos, Wistar, machos (250-300 g), divididos em sete grupos: 1. falso-operado, 2. falso-operado tratado BBG (45 mg/kg i.p.), 3. falso-operado tratado com L-DOPA (30 mg/kg v.o.), 4. animais que receberam a 6-OHDA, denominados de parkinsonianos, 5. parkinsonianos tratados com L-DOPA (30 mg/kg v.o.), 6. parkinsonianos tratados com L-DOPA (30 mg/kg v.o.) e BBG (22,5 mg/kg i.p.) e 7. parkinsonianos tratados com L-DOPA (30 mg/kg v.o.) e BBG (45 mg/kg i.p.). O modelo experimental foi induzido pela injeção de 6-OHDA no corpo estriado dos animais através de cirurgia estereotáxica. Os tratamentos com L-DOPA e BBG iniciaram-se no 15º dia após a cirurgia e foram realizados durante 22 dias. A L-DOPA foi administrada diariamente e o BBG a cada 48 h. A avaliação dos movimentos involuntários anormais (AIMs) foi realizada nos dias 21, 28 e 35 após a cirurgia e a coordenação motora no 36º dia. A degeneração neuronal foi avaliada através da imunomarcação para tirosina hidroxilase e pela coloração por fluoro-jade C. Avaliaou-se alterações no transportador de dopamina, em receptores dopaminérgicos (D1 e D2) e na proteína DARPP-32. O BBG diminuiu os AIMs nos animais parkinsonianos tratados com L-DOPA e melhorou o desempenho de coordenação motora dos animais. A L-DOPA induziu diminuição da imunomarcação para DAT, e da expressão de receptor D1 e de DARPP-32 fosforilada e o BBG reverteu essas alterações. A participação da neuroinflamação também foi avaliada (astrogliose, microgliose, COX-2 e IL-1β). O tratamento com L-DOPA aumentou a neuroinflamação e o foi capaz de BBG diminuir a neuroinflamação. Nossos resultados sugerem que o sistema purinérgico desempenha importante papel no desenvolvimento da discinesia induzida por L-DOPA, e que o BBG possui efeito anti-discinético, possivelmente devido à diminuição da ativação do receptor D1 e da diminuição da neuroinflamação.

Purinerge P2X3-Rezeptoren spielen eine bedeutende Rolle in der Vermittlung chronischer Schmerzen, welche ein führendes Problem des Gesundheitswesens mit vielen sozioökonomischen Konsequenzen darstellen. Die Tatsache, dass P2X3-Rezeptoren fast ausschließlich von nozizeptiven Neuronen exprimiert werden, macht sie trotz ihres besonderen Desensitisierungsverhaltens zu vielversprechenden Angriffspunkten zukünftiger Schmerztherapien, beispielsweise mithilfe kompetitiver Antagonisten an diesen Rezeptoren. Zur Analyse der Wechselwirkungen zwischen Agonist und kompetitivem Antagonist wird meist der Schild-Plot benutzt. Jedoch ist dieser im Falle der sehr schnell desensitisierenden P2X3-Rezeptoren ungeeignet, da die Vorbedingung eines stabilen Gleichgewichts zwischen Agonist und Antagonist aufgrund der Desensitisierung nicht erfüllt ist. Ziel der vorliegenden Arbeit war es, eine neue Methode zur Analyse der Interaktion kompetitiver Antagonisten mit ihrer Bindungsstelle am Beispiel des P2X3-Rezeptors zu entwickeln und so für die Antagonistenbindung bedeutende Aminosäuren der Bindungsstelle zu identifizieren. Mittels der Patch-Clamp-Technik wurden die Effekte der Antagonisten A-317491, TNP-ATP und PPADS auf die vom P2X1,3-Rezeptor-selektiven Agonisten α,β-MeATP induzierten Ströme am P2X3-Wildtyp-Rezeptor und an fünf Rezeptormutanten mit veränderter Ligandenbindungsstelle untersucht. Alle Rezeptoren wurden in HEK293-Zellen exprimiert. Anhand der gemessenen Daten wurde ein Hidden Markov Model (HMM) erstellt, welches die sequentiellen Übergänge des Rezeptors von geschlossen zu offen und desensitisiert in An- und Abwesenheit des Antagonisten miteinander kombiniert. Die am P2X3-Rezeptor induzierten Ströme konnten mithilfe dieses Modells korrekt gefittet und die für die Antagonistenbindung wichtigen Aminosäuren innerhalb der Bindungsstelle bestimmt werden. Als Resultat dieser Arbeit konnte außerdem gezeigt werden, dass das HMM eine geeignete Methode zur Analyse der Wirkung kompetitiver Antagonisten an schnell desensitisierenden Rezeptoren darstellt. Die untersuchten Antagonisten A-317491 und TNP-ATP haben einen kompetitiven Wirkmechanismus, während PPADS eine pseudoirreversible Blockade verursacht.

碩士
國立陽明大學
藥理學研究所
103
Splanchnic angiogenesis in liver cirrhosis with portal hypertension often leads to complications such as gastroesophageal variceal hemorrhage. Even worse, the therapeutic efficacy of vasoconstrictor is adversely affected by poor vasoresponsiveness. Purinergic receptor subtype P2X7 participates in the modulation of inflammation, angiogenesis, fibrogenesis and vascular responsibility, but the relevant influences in cirrhosis remain unknown. In our investigation, Spraque-Dawley rats with/without bile duct ligation (BDL) were treated with brilliant blue G (BBG, P2X7 receptor antagonist and a food additive) from day 15 to day 28 after surgery. Hemodynamics, splanchnic angiogenesis, and liver fibrosis were then evaluated. Additionally, portal-systemic collateral vasoresponsiveness to arginine vasopressin (AVP) was analyzed by pre-treatment with oxidized ATP (oATP, another P2X7 receptor antagonist) in rats receiving BDL surgery 29 days later. Our results revealed that BBG significantly decreased superior mesenteric artery (SMA) flow, severity of portal-systemic shunting, mesenteric window vascular density, and protein expressions of mesenteric angiogenic factors, including vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), phospho-VEGFR2 (p-VEGFR2), platelet-derived growth factor (PDGF), PDGF receptor beta (PDGFRβ), cyclooxygenase (COX)-1, COX-2, and endothelial nitric oxide synthase (eNOS) in BDL rats. In addition, BBG ameliorated liver fibrosis by down-regulating hepatic transforming growth factor beta (TGF-β) and phospho-extracellular signal regulated kinase (p-ERK) expression in BDL rats. The collateral vasocontractility to AVP was significantly enhanced by oATP in BDL rats. Furthermore, oATP also down-regulated eNOS and inducible NOS (iNOS) expressions in splenorenal shunt, the most prominent intra-abdominal shunt vessel in rodents. In conclusion, P2X7 antagonism alleviated splanchnic hyperemia, severity of portal-systemic shunting, mesenteric angiogenesis, liver fibrosis, and improves portal-systemic collateral vasoresponsiveness in cirrhotic rats. Therefore, blockade of P2X7 pathway could be a feasible strategy in treatment of cirrhosis and its complications.