RESUMEN
P2X receptor activation protects in heart failure models. MRS2339 3, a 2-chloro-AMP derivative containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system, activates this cardioprotective channel. Michaelis-Arbuzov and Wittig reactions provided phosphonate analogues of 3, expected to be stable in vivo due to the C-P bond. After chronic administration via a mini-osmotic pump (Alzet), some analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice (genetic model of heart failure) compared to vehicle-infused mice (all inactive at the vasodilatory P2Y(1) receptor). Two phosphonates, (1'S,2'R,3'S,4'R,5'S)-4'-(6-amino-2-chloropurin-9-yl)-2',3'-(dihydroxy)-1'-(phosphonomethylene)-bicyclo[3.1.0]hexane, 4 (MRS2775), and its homologue 9 (MRS2935), both 5'-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening (20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 5'-extended phosphonates, all 2-H analogues, and a CH(3)-phosphonate were inactive. Thus, chronic administration of nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor activation. Thus, we have greatly expanded the range of carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure.
Asunto(s)
Adenosina Monofosfato/farmacología , Cardiotónicos/farmacología , Organofosfonatos/farmacología , Agonistas del Receptor Purinérgico P2 , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Animales , Calcio/metabolismo , Cardiotónicos/administración & dosificación , Cardiotónicos/síntesis química , Línea Celular Tumoral , Ecocardiografía , Femenino , Corazón/efectos de los fármacos , Corazón/fisiopatología , Humanos , Bombas de Infusión , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Ratones , Modelos Químicos , Estructura Molecular , Miocardio/metabolismo , Miocardio/patología , Organofosfonatos/administración & dosificación , Organofosfonatos/química , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y1 , Relación Estructura-ActividadRESUMEN
The P2Y(14) receptor, a nucleotide signaling protein, is activated by uridine-5'-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y(14) agonist. For example, the carboxylate group of uridine-5'-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this strategy retained P2Y(14) activity, and molecular modeling predicted close proximity of this chain to the second extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y(14) potency. For example, the [5'']ribose derivative had an EC(50) of 0.24muM. Selective monofluorination of the glucose moiety indicated a role for the 2''- and 6''-hydroxyl groups of 1 in receptor recognition. The beta-glucoside was twofold less potent than the native alpha-isomer, but methylene replacement of the 1''-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5'-diphosphoglucose also activates the P2Y(2) receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y(14)-selective.
Asunto(s)
Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Relación Estructura-Actividad , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/farmacología , Uridina Difosfato Glucosa/análogos & derivados , Animales , Células COS , Chlorocebus aethiops , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Receptores Purinérgicos P2/química , Fosfolipasas de Tipo C/metabolismo , Nucleótidos de Uracilo/síntesis químicaRESUMEN
Ribose-based nucleoside 5'-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5'-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y(2) receptor but were inactive or weakly active at P2Y(4) receptor. Uridine 5'-(diphospho)phosphonate was approximately as potent at the P2Y(2) receptor as at the UDP-activated P2Y(6) receptor. These results suggest that removal of the 5'-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y(2) receptor. Uridine 5'-(phospho)phosphonate as well as the 5'-methylenephosphonate equivalent of UMP were inactive at the P2Y(4) receptor and exhibited maximal effects at the P2Y(2) receptor that were 50% of that of UTP suggesting novel action of these analogues.
Asunto(s)
Nucleótidos/síntesis química , Agonistas del Receptor Purinérgico P2 , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/síntesis química , Adenosina Difosfato/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/química , Línea Celular Tumoral , Humanos , Nucleótidos/química , Receptores Purinérgicos P2/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/metabolismo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntesis química , Uridina Trifosfato/químicaRESUMEN
INTRODUCTION: Bromine-76-radiolabeled analogues of previously reported high-affinity A(3) adenosine receptor (A(3)AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. METHODS: The radiosyntheses were accomplished by oxidative radiobromination on the N(6)-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. RESULTS: We prepared an agonist ligand {[(76)Br](1'S,2'R,3'S,4'R,5'S)-4'-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581)} in 59% radiochemical yield with a specific activity of 19.5 GBq/micromol and an antagonist ligand {[(76)Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147)} in 65% radiochemical yield with a specific activity of 22 GBq/micromol. The resultant products exhibited the expected high affinity (K(i) approximately 0.6 nM) and specific binding at the human A(3)AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A(3)AR, over a 2-h time course, which suggests the presence of a specific A(3)AR retention mechanism. CONCLUSION: We were able to compare uptake of the [(76)Br]-labeled antagonist MRS5147 to [(76)Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A(3)AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.
Asunto(s)
Radioisótopos de Bromo/química , Nucleósidos/química , Tomografía de Emisión de Positrones/métodos , Receptor de Adenosina A3/metabolismo , Agonistas del Receptor de Adenosina A3 , Antagonistas del Receptor de Adenosina A3 , Animales , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Ligandos , Nucleósidos/metabolismo , Nucleósidos/farmacología , Radioquímica , Ratas , Coloración y Etiquetado , Especificidad por Sustrato , Distribución TisularRESUMEN
A known selective agonist of the A(3) adenosine receptors (AR), MRS1898 [(1'R,2'R,3'S,4'R,5'S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A(3)AR affinity across species, with nanomolar binding at both rat and human A(3)ARs. The radioiodination of MRS1898 on its N (6)-3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [(125)I]MRS1898 bound to the rat A(3)AR with a K(d) value of 0.17 +/- 0.04 nM and a B(max) value of 0.66 +/- 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [(125)I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A(3)AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A(3)AR.
RESUMEN
We have prepared 50-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A3 AR. The H-bonding ability of a group of 9-riboside derivatives containing a 50-uronamide moiety was reduced by modification of the NH; however these derivatives did not display the desired activity as selective A3 AR antagonists, as occurs with 50-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 40-hydroxymethyl group were highly potent and selective antagonists of the human A3 AR. The compounds were synthesized from D-ribose using a reductive free radical decarboxylation of a 50-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A3AR agonists. Compounds 33b-39b (N6-3-halobenzyl and related arylalkyl derivatives) were potent A3AR antagonists with binding Ki values of 0.7-1.4 nM. In a functional assay of [35S]GTPcS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a KB of 8.9 nM. Thus, a highly potent and selective series of A3AR antagonists has been described.
Asunto(s)
Antagonistas del Receptor de Adenosina A3 , Compuestos Bicíclicos con Puentes/química , Nucleósidos/farmacología , Ribosa/química , Algoritmos , Animales , Sitios de Unión , Células CHO/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Nucleósidos/síntesis química , Ensayo de Unión Radioligante , Receptor de Adenosina A3/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Substitution of the ribose moiety of various nucleosides and nucleotides with the (N)-methanocarba ring system increases the potency and selectivity as ligands at certain subtypes of adenosine and P2 receptors. We have prepared a key intermediate in the synthesis of these derivatives, ethyl (1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-hydroxybicyclo[3.1.0]hexane-carboxylate (15), starting from L-ribose (8) as a readily available, enantiopure building block. L-ribose was converted to the corresponding 5'-iodo derivative (9), which was cleaved reductively with Zn. Improvements were made in subsequent steps corresponding to a published route to biologically important (N)-methanocarba 5'-uronamido nucleosides, and new steps were added to prepare related 5'-nucleotides.
Asunto(s)
Adenosina/química , Adenosina/metabolismo , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Nucleósidos/síntesis química , Nucleótidos/síntesis química , Ribosa/química , Adenosina Desaminasa/metabolismo , Compuestos Heterocíclicos con 3 Anillos/química , Ligandos , Nucleósidos/química , Nucleótidos/química , Receptores Purinérgicos P2/metabolismo , EstereoisomerismoRESUMEN
Analogues of the P2X(7) receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized and assayed at the human P2X(7) receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dye (ethidium bromide) in stably transfected HEK293 cells and IL-1beta release in differentiated THP-1 cells. Substitution of the arylsulfonyl moiety with a nitro group increased antagonistic potency relative to methyl substitution, such that compound 21 was slightly more potent than KN-62. Substitution with D-tyrosine in 36 and sterically bulky tyrosyl 2,6-dimethyl groups [corrected] in 9 enhanced antagonistic potency.
Asunto(s)
Antagonistas del Receptor Purinérgico P2 , Tirosina/farmacología , Línea Celular , Humanos , Receptores Purinérgicos P2X7 , Estereoisomerismo , Relación Estructura-Actividad , Tirosina/químicaRESUMEN
P2Y1 is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y1 homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.
Asunto(s)
Modelos Moleculares , Antagonistas del Receptor Purinérgico P2 , Sitios de Unión , Cromatografía Líquida de Alta Presión , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Relación Estructura-Actividad Cuantitativa , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1RESUMEN
Microglia, brain immune cells, engage in the clearance of dead cells or dangerous debris, which is crucial to the maintenance of brain functions. When a neighbouring cell is injured, microglia move rapidly towards it or extend a process to engulf the injured cell. Because cells release or leak ATP when they are stimulated or injured, extracellular nucleotides are thought to be involved in these events. In fact, ATP triggers a dynamic change in the motility of microglia in vitro and in vivo, a previously unrecognized mechanism underlying microglial chemotaxis; in contrast, microglial phagocytosis has received only limited attention. Here we show that microglia express the metabotropic P2Y6 receptor whose activation by endogenous agonist UDP triggers microglial phagocytosis. UDP facilitated the uptake of microspheres in a P2Y6-receptor-dependent manner, which was mimicked by the leakage of endogenous UDP when hippocampal neurons were damaged by kainic acid in vivo and in vitro. In addition, systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in messenger RNA encoding P2Y6 receptors that colocalized with activated microglia were observed. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and could function as a sensor for phagocytosis by sensing diffusible UDP signals, which is a previously unknown pathophysiological function of P2 receptors in microglia.
Asunto(s)
Microglía/efectos de los fármacos , Microglía/inmunología , Fagocitosis/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Uridina Difosfato/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ácido Kaínico/farmacología , Microglía/citología , Microglía/metabolismo , Ratas , Uridina/metabolismo , Uridina Difosfato/metabolismoRESUMEN
P2X purinergic receptors, activated by extracellular ATP, mediate a number of cardiac cellular effects and may be important under pathophysiological conditions. The objective of the present study was to characterize the P2X receptor-mediated ionic current and determine its role in heart failure using the calsequestrin (CSQ) model of cardiomyopathy. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and CSQ mice. The P2X agonist 2-methylthio-ATP (2-meSATP) induced an inward current that was greater in magnitude in CSQ than in WT ventricular cells. The novel agonist, MRS-2339, an N-methanocarba derivative of 2-chloro-AMP relatively resistant to nucleotidase, induced a current in the CSQ myocyte similar to that by 2-meSATP. When administered via a miniosmotic pump (Alzet), it significantly increased longevity compared with vehicle-injected mice (log rank test, P = 0.02). The improvement in survival was associated with decreases in the heart weight-to-body weight ratio and in cardiac myocyte cross-sectional area [MRS-2339-treated mice: 281 +/- 15.4 (SE) mum(2), n = 6 mice vs. vehicle-treated mice: 358 +/- 27.8 mum(2), n = 6 mice, P < 0.05]. MRS-2339 had no vasodilator effect in mouse aorta ring preparations, indicating that its salutary effect in heart failure is not because of any vascular unloading. The cardiac P2X current is upregulated in the CSQ heart failure myocytes. Chronic administration of a nucleotidase-resistant agonist confers a beneficial effect in the CSQ model of heart failure, apparently via an activation of the cardiac P2X receptor. Cardiac P2X receptors represent a novel and potentially important therapeutic target for the treatment of heart failure.
Asunto(s)
Nucleótidos de Adenina/farmacología , Calsecuestrina/metabolismo , Gasto Cardíaco Bajo/prevención & control , Cardiomiopatías/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Agonistas del Receptor Purinérgico P2 , Nucleótidos de Adenina/uso terapéutico , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Bencenosulfonatos/farmacología , Calsecuestrina/genética , Gasto Cardíaco Bajo/etiología , Cardiomiopatías/complicaciones , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X4 , Tionucleótidos/farmacologíaRESUMEN
Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y1 nucleotide receptor and the A3 adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.
Asunto(s)
Nucleósidos/química , Nucleótidos/química , Animales , Cationes , Membrana Celular/metabolismo , Terapia Genética/métodos , Humanos , Ligandos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Purinas/química , Receptor de Adenosina A3/química , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2Y1 , Ribosa/químicaRESUMEN
Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X(2/3)/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X(2/3)/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformationally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4nM at the P2Y1 receptor, with >10000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed.
Asunto(s)
Nucleótidos/química , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Animales , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Nucleótidos/metabolismo , Conformación Proteica , Receptores Purinérgicos P2/metabolismoRESUMEN
INTRODUCTION: Although activation of A3 adenosine receptors attenuates reperfusion lung injury and associated apoptosis, the signaling pathway that mediates this protection remains unclear. Adenosine agonists activate mitogen-activated protein kinases, and these kinases have been implicated in ischemia/reperfusion injury; the purpose of this study was therefore to determine whether A3 adenosine receptor stimulation with reperfusion modulates expression of the different mitogen-activated protein kinases. In addition, we compared the effect of the A3 adenosine agonist IB-MECA with the newly synthesized, highly selective A3 adenosine receptor agonist MRS3558 on injury in reperfused lung. METHOD: Studies were performed in an in vivo spontaneously breathing cat model, in which the left lower lobe of the lung was isolated and subjected to 2 hours of ischemia and 3 hours of reperfusion. The selective A3 adenosine receptor agonists IB-MECA (0.05 mg/kg, 0.1 mg/kg, or 0.3 mg/kg) and MRS3558 (0.05 mg/kg or 0.1 mg/kg) were administered before reperfusion. RESULTS: Both A3 adenosine receptor agonists administered before reperfusion markedly (P < 0.01) attenuated indices of injury and apoptosis, including the percentage of injured alveoli, wet/dry weight ratio, myeloperoxidase activity, TUNEL (in situ TdT-mediated dUTP nick end labeling)-positive cells, and caspase 3 activity and expression. The more pronounced effects at low doses were observed with MRS3558. Increases in phosphorylated c-Jun amino-terminal protein kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2 levels were observed by the end of reperfusion compared with controls. Pretreatment with the A3 agonists upregulated phosphorylated ERK1/2 levels but did not modify phosphorylated JNK and p38 levels. CONCLUSION: The protective effects of A3 adenosine receptor activation are mediated in part through upregulation of phosphorylated ERK. Also, MRS3558 was found to be more potent than IB-MECA in attenuating reperfusion lung injury. The results suggest not only that enhancement of the ERK pathway may shift the balance between cell death and survival toward cell survival, but also that A3 agonists have potential as an effective therapy for ischemia/reperfusion-induced lung injury.
Asunto(s)
Enfermedades Pulmonares/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptor de Adenosina A3/metabolismo , Daño por Reperfusión/metabolismo , Animales , Apoptosis/fisiología , Gatos , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/patología , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/genética , Receptor de Adenosina A3/biosíntesis , Receptor de Adenosina A3/genética , Daño por Reperfusión/genéticaRESUMEN
The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y(2/4) agonist UTPgammaS increased contraction by 52%, similar to beta1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPbetaS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDPbetaS and the UTPgammaS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.
Asunto(s)
Células Musculares/metabolismo , Infarto del Miocardio/metabolismo , Receptores Purinérgicos P2/fisiología , Uridina Difosfato/metabolismo , Uridina Trifosfato/metabolismo , Anciano , Animales , Anticoagulantes/uso terapéutico , Cardiotónicos/farmacología , Dolor en el Pecho/tratamiento farmacológico , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos , Persona de Mediana Edad , Células Musculares/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y2RESUMEN
The highly selective agonists of the A(3) adenosine receptor (AR), Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine), and its 4'-thio analogue, were successfully converted into selective antagonists simply by appending a second N-methyl group on the 5'-uronamide position. The 2-chloro-5'-(N,N-dimethyl)uronamido analogues bound to, but did not activate, the human A(3)AR, with K(i) values of 29 nM (4'-O) and 15 nM (4'-S), showing >100-fold selectivity over A(1), A(2A), and A(2B)ARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-5'-(N,N-dimethyl)uronamido substitution also retained A(3)AR selectivity but lowered affinity.
Asunto(s)
Adenosina/farmacología , Amidas/química , Furanos/química , Receptor de Adenosina A3/química , Ribosa/química , Adenosina/química , Agonistas del Receptor de Adenosina A3 , Antagonistas del Receptor de Adenosina A3 , Animales , Unión Competitiva , Células CHO , Cricetinae , Humanos , Relación Estructura-ActividadRESUMEN
9-(ß-D-Ribosfuranosyluronamide)adenine derivatives that are selective agonists and antagonists of the A3 adenosine receptor (AR) have been derivatized as prodrugs for in vivo delivery. The free hydroxy groups at the 2' and 3' positions of the agonists 2-chloro-N 6-(3-iodobenzyl)-9-(N-methyl-(ß-D-ribosfuranosyluronamide)adenine 2b, the corresponding 4'-thio nucleoside 2c, and antagonists 4a and 4b (5'-N,N-dimethylamides related to 2b and 2c, respectively) were derivatized through simple acylation reactions. The prodrug derivatives were tested in radioligand binding assays at ARs and in a functional assay of adenylate cyclase at the A3AR and found to be considerably less active than the parent drugs. The hydrolysis of nucleoside 2',3'-diesters to regenerate the parent compound in the presence of human blood was demonstrated. 2',3'-Dipropionate esters of 2b and 4a were readily cleaved in a two-step reaction to regenerate the parent drug, on a time scale of two hours. The cleavage of a 2',3'-dihexanoate ester occurred at a slower rate. This indicates that the prodrugs are suitable as masked forms of the biologically active A3AR agonists and antagonists for future evaluation in vivo.
RESUMEN
Ring-constrained adenosine analogues have been designed to act as dual agonists at tissue-protective A(1) and A(3) adenosine receptors (ARs). 9-Ribosides transformed into the ring-constrained (N)-methanocarba-2-chloro-5'-uronamides consistently lost affinity at A(1)/A(2A)ARs and gained at A(3)AR. Among 9-riboside derivatives, only N(6)-cyclopentyl and 7-norbornyl moieties were extrapolated for mixed A(1)/A(3) selectivity and rat/human A(3)AR equipotency. Consequently, 2 was balanced in affinity and potency at A(1)/A(3)ARs as envisioned and dramatically protected in an intact heart model of global ischemia and reperfusion.
Asunto(s)
Agonistas del Receptor de Adenosina A1 , Agonistas del Receptor de Adenosina A3 , Adenosina/análogos & derivados , Cardiotónicos/síntesis química , Adenosina/síntesis química , Adenosina/metabolismo , Adenosina/uso terapéutico , Agonistas del Receptor de Adenosina A2 , Animales , Células CHO , Cricetinae , Diseño de Fármacos , Humanos , Ratones , Infarto del Miocardio/prevención & control , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A3/metabolismo , Daño por Reperfusión/prevención & control , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Combining molecular dynamics (MD) in a hydrated phospholipid (DOPC) bilayer, a Monte Carlo search, and synthesis of locked nucleotide analogues, we discovered that the Southern conformation of the ribose is preferred for ligand recognition by the P2Y(6) receptor. 2'-Deoxy-(S)-methanocarbaUDP was found to be a full agonist of the receptor and displayed a 10-fold higher potency than that for the corresponding flexible 2'-deoxyUDP. MD results also suggested a conformational change of the second extracellular loop consequent to agonist binding.
Asunto(s)
Agonistas del Receptor Purinérgico P2 , Uridina Difosfato/análogos & derivados , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Receptores Purinérgicos P2/metabolismo , Células Tumorales Cultivadas , Uridina Difosfato/metabolismoRESUMEN
Selective agonists and antagonists for A3 adenosine receptors (ARs) are being explored for the treatment of a variety of disorders, including brain and heart ischemic conditions, cancer, and rheumatoid arthritis. This review covers both the structure activity relationships of nucleoside agonist ligands and selected antagonists acting at this receptor and the routes of synthesis. Highly selective agonists have been designed, using both empirical approaches and a semi-rational approach based on molecular modeling. The prototypical A3 agonists IB-MECA 10 and the more receptor-subtype-selective Cl-IB-MECA 11, both of which have affinity in binding to the receptor of approximately 1 nM, have been used widely as pharmacological probes in the elucidation of the physiological role of this receptor. In addition to the exploration of the effects of structural modification of the adenine and ribose moieties on A3AR affinity, the effects of these structural changes on the intrinsic efficacy have also been studied in a systematic fashion. Key structural features determining A3AR interaction include the N6-benzyl group, 2-position substitution such as halo, substitution of ribose (e.g., the (N)-methanocarba ring system, various 2'- and 3'-substitutions and 4'-thio substitution of oxygen). Conformational studies of the ribose moiety and its equivalents indicate that the ring oxygen is not required and the North (N) ring conformation is preferred in binding to the A3AR. Using these observations, a series of ring constrained (N)-methanocarba 5'-uronamide derivatives was recently reported to be highly selective A3AR agonists, the most notable amongst them was MRS3558 113 having a Ki value in binding to the human A3 receptor of 0.3 nM.