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Kenneth A Jacobson - One of the best experts on this subject based on the ideXlab platform.
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Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors.
Biochemical pharmacology, 2006Co-Authors: Liaman K. Mamedova, Zhan-guo Gao, Kenneth A JacobsonAbstract:Abstract ADP is the endogenous agonist for both P2Y1 and P2Y12 receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5′-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y1 receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y12 receptor behaved similarly. We demonstrated here that, unlike with the Gq-coupled P2Y1 receptor, activation of the Gi-coupled P2Y12 receptor does not induce apoptosis. Furthermore, activation of the P2Y12 receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor α (TNFα)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y12 receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y1 receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y12 receptor activation by 10 nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100 pM 2MeSADP, activation of the hP2Y12 receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y12 receptor activation. Apyrase did not affect the ability of TNFα to induce apoptosis in hP2Y12-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y1 and P2Y12 receptors and their opposing effects on cell death pathways.
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synthesis of pyridoxal phosphate derivatives with antagonist activity at the p2y13 receptor
Biochemical Pharmacology, 2005Co-Authors: Yongchul Kim, Jean-marie Boeynaems, Liaman K. Mamedova, Jungsun Lee, Katrin Sak, Frederic Marteau, Kenneth A JacobsonAbstract:We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing Gα16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5′-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5′-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5′-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP3) formation with pIC50 values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y1 receptor-mediated 1321N1 astrocytoma cells with IC50 values of >10 and 0.245 μM, respectively. Both analogues were inactive (IC50 > 10 μM) as antagonists of human P2Y12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y13 receptor in comparison to P2Y1 and P2Y12 receptors, while MRS2603 antagonized both P2Y1 and P2Y13 receptors.
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architecture of p2y nucleotide receptors structural comparison based on sequence analysis mutagenesis and homology modeling
Journal of Medicinal Chemistry, 2004Co-Authors: Stefano Costanzi, Liaman K. Mamedova, Zhan-guo Gao, Kenneth A JacobsonAbstract:Human P2Y receptors encompass at least eight subtypes of Class A G protein-coupled receptors (GPCRs), responding to adenine and/or uracil nucleotides. Using a BLAST search against the Homo sapiens subset of the SWISS−PROT and TrEMBL databases, we identified 68 proteins showing high similarity to P2Y receptors. To address the problem of low sequence identity between rhodopsin and the P2Y receptors, we performed a multiple-sequence alignment of the retrieved proteins and the template bovine rhodopsin, combining manual identification of the transmembrane domains (TMs) with automatic techniques. The resulting phylogenetic tree delineated two distinct subgroups of P2Y receptors: Gq-coupled subtypes (e.g., P2Y1) and those coupled to Gi (e.g., P2Y12). On the basis of sequence comparison we mutated three Tyr residues of the putative P2Y1 binding pocket to Ala and Phe and characterized pharmacologically the mutant receptors expressed in COS-7 cells. The mutation of Y306 (7.35, site of a cationic residue in P2Y12)...
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molecular recognition at purine and pyrimidine nucleotide p2 receptors
Current Topics in Medicinal Chemistry, 2004Co-Authors: Kenneth A Jacobson, Stefano Costanzi, Michihiro Ohno, Bhalchandra V Joshi, Pedro Besada, Susanna TchilibonAbstract:In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide / nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-g-S and UDP-b-S are selective agonists for P2Y2 / P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7.
Christa E. Müller - One of the best experts on this subject based on the ideXlab platform.
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Contribution of platelet P2Y12 receptors to chronic Complete Freund's adjuvant‐induced inflammatory pain
Journal of Thrombosis and Haemostasis, 2017Co-Authors: K. Bekő, Bence Koványi, Gergely Horváth, Adam Denes, Zsuzsanna Környei, Bálint Botz, Zsuzsanna Helyes, Christa E. Müller, Flóra Gölöncsér, Beata SperlaghAbstract:Essentials The role of platelet P2Y12 receptors in the regulation of chronic inflammatory pain is unknown. Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain model was used in mice. Gene deficiency and antagonists of P2Y12 receptors attenuate hyperalgesia and local inflammation. Platelet P2Y12 receptors contribute to these effects in the chronic phase of inflammation. SummaryBackground P2Y12 receptor antagonists are widely used in clinical practice to inhibit platelet aggregation. P2Y12 receptors are also known to regulate different forms of pain as well as local and systemic inflammation. However, it is not known whether platelet P2Y12 receptors contribute to these effects. Objectives To explore the contribution of platelet P2Y12 receptors to chronic inflammatory pain in mice. Methods Complete Freund's adjuvant (CFA)-induced chronic inflammatory pain was induced in wild-type and P2ry12 gene-deficient (P2ry12−/−) mice, and the potent, direct-acting and reversible P2Y12 receptor antagonists PSB-0739 and cangrelor were used. Results CFA-induced mechanical hyperalgesia was significantly decreased in P2ry12−/− mice for up to 14 days, and increased neutrophil myeloperoxidase activity and tumor necrosis factor (TNF)-α and CXCL1 (KC) levels in the hind paws were also attenuated in the acute inflammation phase. At day 14, increased interleukin (IL)-1β, IL-6, TNF-α and KC levels were attenuated in P2ry12−/− mice. PSB-0739 and cangrelor reversed hyperalgesia in wild-type mice but had no effect in P2ry12−/− mice, and PSB-0739 was also effective when applied locally. The effects of both local and systemic PSB-0739 were prevented by A-803467, a selective NaV1.8 channel antagonist, suggesting the involvement of NaV1.8 channels in the antihyperalgesic effect. Platelet depletion by anti-mouse CD41 antibody decreased hyperalgesia and attenuated the proinflammatory cytokine response in wild-type but not in P2ry12–/– mice on day 14. Conclusions In conclusion, P2Y12 receptors regulate CFA-induced hyperalgesia and the local inflammatory response, and platelet P2Y12 receptors contribute to these effects in the chronic inflammation phase.
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structure of the human P2Y12 receptor in complex with an antithrombotic drug
Nature, 2014Co-Authors: Kaihua Zhang, Jin Zhang, Dandan Zhang, Steven M Moss, Silvia Paoletta, Evgeny Kiselev, Weizhen Lu, Gustavo Fenalti, Wenru Zhang, Christa E. MüllerAbstract:The X-ray crystal structure of the human P2Y12 receptor, which regulates platelet activation and thrombus formation, is solved in complex with an antithrombotic drug, providing insights for the development of new drugs. Two papers in this issue of Nature present the crystal structures of the human P2Y12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors1. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo2, which limits our understanding of this receptor family. P2Y12R regulates platelet activation and thrombus formation3,4, and several antithrombotic drugs targeting P2Y12R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor)5,6 suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors7,8. Here we report the 2.6 A resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.
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agonist bound structure of the human P2Y12 receptor
Nature, 2014Co-Authors: Jin Zhang, Christa E. Müller, Kaihua Zhang, Dandan Zhang, Silvia Paoletta, Wenru Zhang, Tingting Li, Huaiyu Yang, Hualiang Jiang, Vadim CherezovAbstract:An X-ray structure of human P2Y12 receptor, a clinical drug target for platelet aggregation inhibitors, is presented in complex with an agonist, providing insight into the δ-group of class A G-protein-coupled receptors. Two papers in this issue of Nature present the crystal structures of the human P2Y12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y12R provided useful insights into ligand binding1,2,3,4, the agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here we report the structures of the human P2Y12R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 A resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 A resolution. These structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing questions surrounding P2Y12R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.
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P2 Receptors Activated by Uracil Nucleotides - An Update
Current Medicinal Chemistry, 2006Co-Authors: Andreas Brunschweiger, Christa E. MüllerAbstract:Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors (GPCRs) of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y2, P2Y4, P2Y6 and P2Y14. P2Y2 and P2Y4 receptors are activated by UTP (the P2Y2, and the rat but not the human P2Y4 receptor are also activated by ATP), the P2Y6 receptor is activated by UDP, and the P2Y14 receptor by UDP-glucose. Furthermore, non-P2Y GPCRs, the cysteinylleukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinylleukotrienes. While P2Y2, P2Y4, and P2Y6 receptor activation results in stimulation of phospholipase C, the P2Y14 receptor is coupled to inhibition of adenylate cyclase. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. The P2Y2 receptor agonists diuridine tetraphosphate (diquafosol) and the uracil-cytosine dinucleotide denufosol are currently undergoing clinical trials for dry eye disease, retinal detachment disease, upper respiratory tract symptoms, and cystic fibrosis, respectively. The first antagonists for P2Y2 and P2Y6 receptors that appear to be selective versus other P2Y receptor subtypes have recently been described. Selective antagonists for P2Y4 and P2Y14 receptors are still lacking. Uracil nucleotide-sensitive P2Y receptor subtypes may constitute future targets for the treatment of certain cancer types, vascular diseases, inflammatory diseases, and immunomodulatory intervention. They have also been proposed to play a role in neurodegenerative diseases. This article is an updated version of "P2-Pyrimidinergic Receptors and Their Ligands" by C. E. Muller published in Curr. Pharm. Des. 2002, 8, 2353-2369.
Marco Cattaneo - One of the best experts on this subject based on the ideXlab platform.
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inhibition of the platelet P2Y12 receptor for adenosine diphosphate does not impair the capacity of platelet to synthesize thromboxane a2
European Heart Journal, 2016Co-Authors: Mariangela Scavone, Eti Alessandra Femia, Vera Caroppo, Marco CattaneoAbstract:Aims Patients with acute coronary syndromes (ACSs) are treated with acetylsalicylic acid (ASA) and antagonists of the P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP). Based on the demonstration that P2Y12R antagonists inhibit thromboxane A2 (TxA2) production (target of ASA), it was surmised that ACS patients might be treated with P2Y12R antagonists only. However, this demonstration contrasts with the results of previous studies. The aim of this study was to test whether P2Y12R antagonists have off-target/indirect inhibitory effects on platelet TxA2 production. Methods and results We studied 3 patients with inherited P2Y12R deficiency and 33 healthy subjects. Serum TxB2 (TxA2 metabolite) levels were similar in P2Y12R-deficient patients and healthy subjects and were not decreased by P2Y12R antagonists in vitro . Serum TxB2 levels did not decrease in 20 patients treated with prasugrel (10 mg q.i.d.) or placebo for 14 days. Arachidonic acid- and collagen-induced platelet aggregation (PA) and TxB2 production in platelet-rich plasma (PRP) of healthy subjects were inhibited in vitro by P2Y12R antagonists. However, P2Y12R antagonists did not inhibit TxB2 production when PA was prevented by avoiding the stirring of PRP in the aggregometer. The P2Y1 ADP-receptor antagonist MRS2500 had similar effects on PA and TxB2 production as P2Y12R antagonists. Acetylsalicylic acid inhibited TxB2 production more effectively than a P2Y12R antagonist; only the combination of ASA and a P2Y12R antagonist inhibited PA induced by high concentration of collagen. Conclusion Inherited deficiency or pharmacological inhibition of P2Y12R does not affect the platelet capacity to synthesize TxA2. There is no pharmacological evidence that ACS patients may be safely treated with P2Y12R antagonists without ASA.
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Constitutively released adenosine diphosphate regulates proplatelet formation by human megakaryocytes.
Haematologica, 2012Co-Authors: Alessandra Balduini, Christian A. Di Buduo, Alessandro Malara, Anna Lecchi, Paola Rebuzzini, Manuela Currao, Isabella Pallotta, Joseph A. Jakubowski, Marco CattaneoAbstract:Background The interaction of adenosine diphosphate with its P2Y1 and P2Y12 receptors on platelets is important for platelet function. However, nothing is known about adenosine diphosphate and its function in human megakaryocytes. Design and Methods We studied the role of adenosine diphosphate and P2Y receptors on proplatelet formation by human megakaryocytes in culture. Results Megakaryocytes expressed all the known eight subtypes of P2Y receptors, and constitutively released adenosine diphosphate. Proplatelet formation was inhibited by the adenosine diphosphate scavengers apyrase and CP/CPK by 60-70% and by the P2Y12 inhibitors cangrelor and 2-MeSAMP by 50-60%, but was not inhibited by the P2Y1 inhibitor MRS 2179. However, the active metabolites of the anti-P2Y12 drugs, clopidogrel and prasugrel, did not inhibit proplatelet formation. Since cangrelor and 2-MeSAMP also interact with P2Y13, we hypothesized that P2Y13, rather than P2Y12 is involved in adenosine diphosphate-regulated proplatelet formation. The specific P2Y13 inhibitor MRS 2211 inhibited proplatelet formation in a concentration-dependent manner. Megakaryocytes from a patient with severe congenital P2Y12 deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. Conclusions This is the first demonstration that adenosine diphosphate released by megakaryocytes regulates their function by interacting with P2Y13. The clinical relevance of this not previously described physiological role of adenosine diphosphate and P2Y13 requires further exploration.
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New P2Y12 Inhibitors
Circulation, 2010Co-Authors: Marco CattaneoAbstract:A denosine diphosphate (ADP) plays a key role in the genesis of physiological platelet-rich hemostatic plugs and of pathological arterial thrombi.1 The transduction of the ADP signal involves its interaction with 2 platelet receptors, the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor, which belong to the family of purinergic P2 receptors. Concomitant activation of both the Gq and Gi pathways by ADP is necessary to elicit normal platelet aggregation.2 In addition to its role in ADP-induced platelet aggregation, P2Y12 (Figure 1) also mediates the potentiation of platelet secretion induced by strong agonists, which is independent of the formation of large aggregates and thromboxane A2 synthesis2; the stabilization of thrombin-induced platelet aggregates2; shear-induced platelet aggregation2; and the inhibition of the antiplatelet effects of the natural regulator of platelet function, prostacyclin.3 In contrast to P2Y1, P2Y12 has a very selective tissue distribution, making it an attractive molecular target for therapeutic intervention. Indeed, P2Y12 is the target of efficacious antithrombotic agents.1 Figure 1. Central role of P2Y12 in platelet aggregation. ADP, by interacting with P2Y12, a 7-transmembrane receptor that is coupled to the inhibitory G protein Gi, induces platelet aggregation and amplifies the aggregation response that is induced by other agonists or by ADP itself, interacting with its other platelet receptor, P2Y1. In addition, P2Y12 stabilizes the platelet aggregates and amplifies the secretion of platelet dense granules stimulated by secretion-inducing agonists (which are coupled to Gq). Although P2Y12 is coupled to inhibition of adenylyl cyclase (AC) through Gi, this function does not appear to be directly related to P2Y12-mediated platelet activation. However, it could have important implications in vivo, …
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P2Y12 receptor antagonists: a rapidly expanding group of antiplatelet agents.
European heart journal, 2006Co-Authors: Marco CattaneoAbstract:Adenosine-5′-diphosphate (ADP) plays a key role in platelet function, because, although ADP itself is a weak platelet agonist, when secreted from the platelet dense granules where it is stored, it amplifies the platelet responses induced by other platelet agonists.1 The transduction of the ADP signal involves both a transient rise in free cytoplasmic calcium mediated by the Gq-coupled P2Y1 receptor, and inhibition of adenylyl cyclase mediated by the Gi-coupled P2Y12 receptor. Concomitant activation of both the Gq and Gi pathways by ADP is necessary to elicit normal ADP-induced platelet aggregation. Activation of the Gq pathway through P2Y1 leads to platelet shape change and rapidly reversible aggregation, whereas the activation of the Gi pathway through P2Y12 elicits a slow progressive and sustained platelet aggregation not preceded by shape change. In addition to its role in ADP-induced platelet aggregation, P2Y12 mediates the potentiation of platelet secretion induced by strong agonists and the stabilization of thrombin-induced platelet aggregates.1 P2Y12 has a more selective tissue distribution than P2Y1, making it an attractive molecular target for therapeutic intervention. Indeed, P2Y12 is the target of efficacious antithrombotic agents like ticlopidine and clopidogrel, which are already used in clinical practice either alone or in combination with other antithrombotic drugs.2 Ticlopidine and clopidogrel are structurally related compounds, belonging to the thienopyridine family of ADP receptor antagonists; they are pro-drugs that are inactive in vitro and need to be metabolized in vivo by the hepatic cytochrome P-450 1A enzymatic pathway to active metabolites, which have very short half-lives. They irreversibly and specifically inhibit the function of the platelet P2Y12 receptor, reproducing the platelet function abnormalities that are observed in patients who are congenitally deficient in P2Y12 and … *Corresponding author. Tel/fax: +39 0250323095. E-mail address : marco.cattaneo{at}unimi.it
David Erlinge - One of the best experts on this subject based on the ideXlab platform.
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assessment of p2y 12 inhibition with the point of care device verifynow P2Y12 in patients treated with prasugrel or clopidogrel coadministered with aspirin
American Heart Journal, 2009Co-Authors: Christoph Varenhorst, Joseph A. Jakubowski, Oscar O Braun, David Erlinge, Stefan James, John T Brandt, Kenneth J Winters, Sylvia Olofsson, Lars Wallentin, Agneta SiegbahnAbstract:Background Variability in response to thienopyridines has led to the development of point-of-care devices to assess adenosine diphosphate (ADP)-induced platelet aggregation. These tests need to be evaluated in comparison to reference measurements of P2Y(12) function during different thienopyridine treatments. Methods After a run-in on 75 mg aspirin, I 10 subjects were randomized to double-blind treatment with clopidogrel 600 mg loading dose (LD)/75 mg maintenance dose (MD) or prasugrel 60 mg LD/10 mg MD. Antiplatelet effects were evaluated by VerifyNow P2Y12 (VN-P2Y12) device (Accumetrics, San Diego, CA), vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay, and light transmission aggregometry (LTA). Prasugrel's and clopidogrel's active metabolite concentration were also determined. Results Dose- and time-dependent inhibition of P2Y(12) was evident with VN-P2Y12. There was strong correlation with VN-P2Y12 and VASP or LTA for all treatments through a wide range of P2Y(12) function. At high levels of P2Y(12) inhibition, platelet function measured by VN-P2Y12 was maximally inhibited and could not reflect further changes seen with VASP or LTA methods. Correlation was also observed between exposure to clopidogrel's active metabolite and VN-F`2Y12 during MD and LD, whereas it was observed only with prasugrel MD. Conclusion The VN-P2Y12 correlated strongly with inhibition of P2Y(12) function, as measured with either VASP or LTA. VN-P2Y12 also correlated to exposure to the active metabolite of prasugrel and clopidogrel up to levels associated with assumed saturation of the P2Y(12) receptor. (Am Heart J 2009; 1 57:562.e1-562.e9.) (Less)
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adp receptor P2Y12 is expressed in vascular smooth muscle cells and stimulates contraction in human blood vessels
Arteriosclerosis Thrombosis and Vascular Biology, 2004Co-Authors: Annakarin Wihlborg, Lingwei Wang, Oscar O Braun, Atli Eyjolfsson, Ronny Gustafsson, Tomas Gudbjartsson, David ErlingeAbstract:Objective— ADP plays an important role in platelet aggregation by activating P2Y12 receptors. We assessed the hypothesis that P2Y12 receptors are expressed in vascular smooth muscle cells (VSMC). Methods and Results— P2Y12 receptor mRNA was found to have a high expression among the P2 receptors in human VSMC, significantly higher than the other 2 ADP receptors (P2Y1 and P2Y13, real-time polymerase chain reaction). Western blots gave a band of 50 kD, similar to that in platelets. To unmask a P2Y12 receptor-mediated vasoconstriction by simulating the in vivo situation, vessels were precontracted to a submaximal level. 2-MeSADP stimulated contractions in vessel segments from internal mammary artery (IM), IM branches and small veins (Emax=15±6% of 60mmol/L K+ contraction, pEC50=5.6±0.6, Emax=21±1%, pEC50=6.8±0.1, and Emax=48±9%, pEC50=6.6±0.4). The selective P2Y12 antagonist AR-C67085 blocked 2-MeSADP contractions. The contraction was not reduced in patients using clopidogrel, a drug inhibiting ADP-induced pl...
Jin Zhang - One of the best experts on this subject based on the ideXlab platform.
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agonist bound structure of the human P2Y12 receptor
Nature, 2014Co-Authors: Jin Zhang, Christa E. Müller, Kaihua Zhang, Dandan Zhang, Silvia Paoletta, Wenru Zhang, Tingting Li, Huaiyu Yang, Hualiang Jiang, Vadim CherezovAbstract:An X-ray structure of human P2Y12 receptor, a clinical drug target for platelet aggregation inhibitors, is presented in complex with an agonist, providing insight into the δ-group of class A G-protein-coupled receptors. Two papers in this issue of Nature present the crystal structures of the human P2Y12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y12R provided useful insights into ligand binding1,2,3,4, the agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here we report the structures of the human P2Y12R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 A resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 A resolution. These structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing questions surrounding P2Y12R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.
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structure of the human P2Y12 receptor in complex with an antithrombotic drug
Nature, 2014Co-Authors: Kaihua Zhang, Jin Zhang, Dandan Zhang, Steven M Moss, Silvia Paoletta, Evgeny Kiselev, Weizhen Lu, Gustavo Fenalti, Wenru Zhang, Christa E. MüllerAbstract:The X-ray crystal structure of the human P2Y12 receptor, which regulates platelet activation and thrombus formation, is solved in complex with an antithrombotic drug, providing insights for the development of new drugs. Two papers in this issue of Nature present the crystal structures of the human P2Y12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors1. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo2, which limits our understanding of this receptor family. P2Y12R regulates platelet activation and thrombus formation3,4, and several antithrombotic drugs targeting P2Y12R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor)5,6 suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors7,8. Here we report the 2.6 A resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.
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adenosine diphosphate adp induced thromboxane a2generation in human platelets requires coordinated signaling through integrin αiibβ3 and adp receptors
Blood, 2002Co-Authors: Jianguo Jin, Todd M. Quinton, Jin Zhang, Susan E Rittenhouse, Satya P. KunapuliAbstract:Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A2, another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-βγ-dichloromethylene adenosine 5′-triphosphate (AR-C67085), a P2Y12 receptor–selective antagonist, and adenosine-2′-phosphate-5′-phosphate (A2P5P), a P2Y1 receptor–selective antagonist, inhibited ADP-induced thromboxane A2 generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A2 production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid–induced platelet aggregation, it failed to inhibit thromboxane A2 generation induced by arachidonic acid. Thus, ADP-induced arachidonic acid liberation, but not subsequent conversion to thromboxane A2, requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site–6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin αIIbβ3, caused both platelet aggregation and thromboxane A2 generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A2 generation, indicating that these signaling molecules have no significant role in phospholipase A2 activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A2, suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A2 activation, resulting in arachidonic acid liberation and thromboxane A2 generation.
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adenosine diphosphate adp induced thromboxane a2generation in human platelets requires coordinated signaling through integrin αiibβ3 and adp receptors
Blood, 2002Co-Authors: Jianguo Jin, Todd M. Quinton, Jin Zhang, Susan E Rittenhouse, Satya P. KunapuliAbstract:Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A(2), another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-beta gamma-dichloromethylene adenosine 5'-triphosphate (AR-C67085), a P2Y12 receptor-selective antagonist, and adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, inhibited ADP-induced thromboxane A(2) generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A(2) production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid-induced platelet aggregation, it failed to inhibit thromboxane A(2) generation induced by arachidonic acid. Thus, ADP-induced arachidonic acid liberation, but not subsequent conversion to thromboxane A(2), requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site-6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin alpha(IIb)beta(3), caused both platelet aggregation and thromboxane A(2) generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A(2) generation, indicating that these signaling molecules have no significant role in phospholipase A(2) activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A(2), suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A(2) activation, resulting in arachidonic acid liberation and thromboxane A(2) generation.
