Molecular interaction of HIC, an agonist of P2Y1 receptor, and its role in prostate cancer apoptosis.

Prostate cancer is a heterogeneous, slow growing asymptomatic cancer that predominantly affects man. A purinergic G-protein coupled receptor, P2Y1R, is targeted for its therapeutic value since it plays a crucial role in many key molecular events of cancer progression and invasion. Our previous study demonstrated that indoline derivative, 1 ((1-(2-Hydroxy-5-nitrophenyl) (4-hydroxyphenyl) methyl)indoline-4­carbonitrile; HIC), stimulates prostate cancer cell (PCa) growth inhibition via P2Y1R. However, the mode of interaction of P2Y1R with HIC involved in this process remains unclear. Here, we have reported the molecular interactions of HIC with P2Y1R. Molecular dynamics simulation was performed that revealed the stable specific binding of the protein-ligand complex. In vitro analysis has shown increased apoptosis of PCa-cells, PC3, and DU145, upon specific interaction of P2Y1R-HIC. This was further validated using siRNA analysis that showed a higher percentage of apoptotic cells in PCa-cells transfected with P2Y-siRNA-MRS2365 than P2Y-siRNA-HIC treatment. Decreased mitochondrial membrane potential (MMP) activity and reduced glutathione (GSH) level show their role in P2Y1R-HIC mediated apoptosis. These in silico and in vitro results confirmed that HIC could induce mitochondrial apoptotic signaling through the P2Y1R activation. Thus, HIC being a potential ligand upon interaction with P2Y1R might have therapeutic value for the treatment of prostate cancer.

Switching a Xanthine Oxidase Inhibitor to a Dual-Target Antagonist of P2Y1 and P2Y12 as an Oral Antiplatelet Agent with a Wider Therapeutic Window in Rats than Ticagrelor.

ADP-mediated platelet aggregation is signaled through G protein-coupled receptors P2Y1 and P2Y12 on the platelet. The clinical effectiveness of inhibiting P2Y12 has been well established, and preclinical studies indicated that the inhibition of P2Y1 could provide equivalent antithrombotic efficacy as P2Y12 antagonists and reduce bleeding risks. On the basis of the 2-phenyl-1H-imidazole scaffold of our previously reported xanthine oxidase inhibitor WSJ-557, we first achieved the transition from the xanthine oxidase inhibitors to dual-target antagonists against P2Y1 and P2Y12. We described the structure-activity relationships of the 2-phenyl-1H-imidazole compounds, which led to the identification of the most potent antiplatelet agents, 24w and 25w, both showing a rapid onset of action in pharmacokinetic study. Furthermore, the rat model suggested that 24w demonstrated a wider therapeutic window than ticagrelor, displaying equivalent and dose-dependent antithrombotic efficacy with lower blood loss compared to ticagrelor at same oral dose. These results supported that 24w and 25w could be promising drug candidates.

Synthesis and preclinical validation of novel P2Y1 receptor ligands as a potent anti-prostate cancer agent.

Purinergic receptor is a potential drug target for neuropathic pain, Alzheimer disease, and prostate cancer. Focusing on the structure-based ligand discovery, docking analysis on the crystal structure of P2Y1 receptor (P2Y1R) with 923 derivatives of 1-indolinoalkyl 2-phenolic compound is performed to understand the molecular insights of the receptor. The structural model identified the top novel ligands, 426 (compound 1) and 636 (compound 2) having highest binding affinity with the docking score of -7.38 and -6.92. We have reported the interaction efficacy and the dynamics of P2Y1R protein with the ligands. The best hits synthesized were experimentally optimized as a potent P2Y1 agonists. These ligands exhibits anti-proliferative effect against the PC-3 and DU-145 cells (IC50 = 15 µM - 33 µM) with significant increase in the calcium level in dose- and time-dependent manner. Moreover, the activation of P2Y1R induced the apoptosis via Capase3/7 and ROS signaling pathway. Thus it is evidenced that the newly synthesized ligands, as a P2Y1R agonists could potentially act as a therapeutic drug for treating prostate cancer.

Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

Salvianolic acids from antithrombotic Traditional Chinese Medicine Danshen are antagonists of human P2Y1 and P2Y12 receptors.

Many hemorheologic Traditional Chinese Medicines (TCMs) that are widely-used clinically lack molecular mechanisms of action. We hypothesized that some of the active components of hemorheologic TCMs may function through targeting prothrombotic P2Y1 and/or P2Y12 receptors. The interactions between 253 antithrombotic compounds from TCM and these two G protein-coupled P2Y receptors were evaluated using virtual screening. Eleven highly ranked hits were further tested in radioligand binding and functional assays. Among these compounds, salvianolic acid A and C antagonized the activity of both P2Y1 and P2Y12 receptors in the low µM range, while salvianolic acid B antagonized the P2Y12 receptor. These three salvianolic acids are the major active components of the broadly-used hemorheologic TCM Danshen (Salvia militorrhiza), the antithrombotic molecular mechanisms of which were largely unknown. Thus, the combination of virtual screening and experimental validation identified potential mechanisms of action of multicomponent drugs that are already employed clinically.

The Molecular Mechanism Underlying Ligand Binding to the Membrane-Embedded Site of a G-Protein-Coupled Receptor.

The crystal structure of P2Y1 receptor (P2Y1R), a class A GPCR, revealed a special extra-helical site for its antagonist, BPTU, which locates in-between the membrane and the protein. However, due to the limitation of crystallization experiments, the membrane was mimicked by use of detergents, and the information related to the binding of BPTU to the receptor in the membrane environment is rather limited. In the present work, we conducted a total of ∼7.5 µs all-atom simulations in explicit solvent using conventional molecular dynamics and multiple enhanced sampling methods, with models of BPTU and a POPC bilayer, both in the absence and presence of P2Y1R. Our simulations revealed that BPTU prefers partitioning into the interface of polar/lipophilic region of the lipid bilayer before associating with the receptor. Then, it interacts with the second extracellular loop of the receptor and reaches the binding site through the lipid-receptor interface. In addition, by use of funnel-metadynamics simulations which efficiently enhance the sampling of bound and unbound states, we provide a statistically accurate description of the underlying binding free energy landscape. The calculated absolute ligand-receptor binding affinity is in excellent agreement with the experimental data (Δ Gb0_theo = -11.5 kcal mol-1, Δ Gb0_exp= -11.7 kcal mol-1). Our study broadens the view of the current experimental/theoretical models and our understanding of the protein-ligand recognition mechanism in the lipid environment. The strategy used in this work is potentially applicable to investigate ligands association/dissociation with other membrane-embedded sites, allowing identification of compounds targeting membrane receptors of pharmacological interest.

Diverse signalling of the platelet P2Y1 receptor leads to a dichotomy in platelet function.

Platelet P2Y1 receptor signalling via RhoGTPases is necessary for platelet-dependent leukocyte recruitment, where no platelet aggregation is observed. We investigated signalling cascades involved in distinct P2Y1-dependent platelet activities in vitro, using specific inhibitors for phospholipase C (PLC) (U73122, to inhibit the canonical pathway), and RhoGTPases: Rac1 (NSC23766) and RhoA (ROCK inhibitor GSK429286). Human platelet rich plasma (for platelet aggregation) or isolated washed platelets (for chemotaxis assays) was treated with U73122, GSK429286 or NSC23766 prior to stimulation with adenosine diphosphate (ADP) or the P2Y1 specific agonist MRS2365. Aggregation, chemotaxis (towards f-MLP), or platelet-induced human neutrophil chemotaxis (PINC) towards macrophage derived chemokine (MDC) was assessed. Molecular docking of ADP and MRS2365 to P2Y1 was analysed using AutoDock Smina followed by GOLD molecular docking in the Accelrys Discovery Studio software. Inhibition of PLC, but not Rac1 or RhoA, suppressed platelet aggregation induced by ADP and MRS2365. In contrast, platelet chemotaxis and PINC, were significantly attenuated by inhibition of platelet Rac1 or RhoA, but not PLC. MRS2365, compared to ADP had a less pronounced effect on P2Y1-induced aggregation, but a similar efficacy to stimulate platelet chemotaxis and PINC, which might be explained by differences in molecular interaction of ADP compared to MRS2365 with the P2Y1 receptor. Platelet P2Y1 receptor activation during inflammation signals through alternate pathways involving Rho GTPases in contrast to canonical P2Y1 receptor induced PLC signalling. This might be explained by selective molecular interactions of ligands within the orthosteric site of the P2Y1 receptor.

Demystifying P2Y1 Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses.

We performed a molecular modeling analysis of 100 nucleotide-like bisphosphates and 46 non-nucleotide arylurea derivatives previously reported as P2Y1R binders using the recently solved hP2Y1R structures. We initially docked the compounds at the X-ray structures and identified the binding modes of representative compounds highlighting key patterns in the structure-activity relationship (SAR). We subsequently subjected receptor complexes with selected key agonists (2MeSADP and MRS2268) and antagonists (MRS2500 and BPTU) to membrane molecular dynamics (MD) simulations (at least 200 ns run in triplicate, simulation time 0.6-1.6 µs per ligand system) while considering alternative protonation states of nucleotides. Comparing the temporal evolution of the ligand-protein interaction patterns with available site-directed mutagenesis (SDM) data and P2Y1R apo state simulation provided further SAR insights and suggested reasonable explanations for loss/gain of binding affinity as well as the most relevant charged species for nucleotide ligands. The MD analysis also predicted local conformational changes required for the receptor inactive state to accommodate nucleotide agonists.

Identification of a Different Agonist-Binding Site and Activation Mechanism of the Human P2Y1 Receptor.

The human P2Y1 receptor (P2Y1R) is a purinergic G-protein-coupled receptor (GPCR) that functions as a receptor for adenosine 5'-diphosphate (ADP). An antagonist of P2Y1R might potentially have antithrombotic effects, whereas agonists might serve as antidiabetic agents. On the basis of the antagonist-bound MRS2500-P2Y1R crystal structure, we constructed computational models of apo-P2Y1R and the agonist-receptor complex 2MeSADP-P2Y1R. We then performed conventional molecular dynamics (cMD) and accelerated molecular dynamics (aMD) simulations to study the conformational dynamics after binding with agonist/antagonist as well as the P2Y1R activation mechanism. We identified a new agonist-binding site of P2Y1R that is consistent with previous mutagenesis data. This new site is deeper than those of the agonist ADP in the recently simulated ADP-P2Y1R structure and the antagonist MRS2500 in the MRS2500-P2Y1R crystal structure. During P2Y1R activation, the cytoplasmic end of helix VI shifts outward 9.1 Å, the Ser1463.47-Tyr2375.58 hydrogen bond breaks, a Tyr2375.58-Val2626.37 hydrogen bond forms, and the conformation of the χ1 rotamer of Phe2696.44 changes from parallel to perpendicular to helix VI. The apo-P2Y1R system and the MRS2500-P2Y1R system remain inactive. The newly identified agonist binding site and activation mechanism revealed in this study may aid in the design of P2Y1R antagonists/agonists as antithrombotic/antidiabetic agents, respectively.

Distinct Signaling Patterns of Allosteric Antagonism at the P2Y1 Receptor.

Traditionally, G protein-coupled receptor antagonists are classified as competitive or noncompetitive and surmountable or insurmountable based on functional antagonism. P2Y1 receptor (P2Y1R) structures showed two antagonists binding to two spatially distinct sites: nucleotide MRS2500 (orthosteric, contacting the helical bundle) and urea BPTU (allosteric, on the external receptor surface). However, the nature of their P2Y1R antagonism has not been characterized. Here we characterized BPTU antagonism at various signaling pathways activated by structurally diverse agonists. BPTU rightward shifted the concentration-response curves of both 2-methylthioadenosine 5'-diphosphate trisodium salt and MRS2365 (5'-diphosphates) in some signaling events, such as extracellular signal-regulated kinase 1/2 and label free, in a parallel manner without affecting the maximum agonist effect (Emax) but antagonized insurmountably (suppressed agonist Emax) in signaling events such as guanosine 5'-3-O-(thio)triphosphate binding and ß-arrestin2 recruitment. However, with dinucleotide Ap4A as an agonist, BPTU suppressed the Emax insurmountably in all signaling pathways. By comparison, MRS2500 behaved as surmountable antagonist rightward-shifting concentration-response curves of all three agonists in a parallel manner for all signaling pathways measured. Thus, we demonstrated a previously undocumented phenomenon that P2Y1R antagonism patterns could vary in different signaling pathways, which could be related to conformational selection, signaling amplification, and probe dependence. This phenomenon may apply generally to other receptors considering that antagonism by a specific ligand is often not compared at multiple signaling pathways. Thus, antagonism can be surmountable or insurmountable depending on the signaling pathways measured and the agonists used, which should be of broad relevance to drug discovery and disease treatment.

Ligand binding to a G protein-coupled receptor captured in a mass spectrometer.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors belong to the largest family of membrane-embedded cell surface proteins and are involved in a diverse array of physiological processes. Despite progress in the mass spectrometry of membrane protein complexes, G protein-coupled receptors have remained intractable because of their low yield and instability after extraction from cell membranes. We established conditions in the mass spectrometer that preserve noncovalent ligand binding to the human purinergic receptor P2Y1. Results established differing affinities for nucleotides and the drug MRS2500 and link antagonist binding with the absence of receptor phosphorylation. Overall, therefore, our results are consistent with drug binding, preventing the conformational changes that facilitate downstream signaling. More generally, we highlight opportunities for mass spectrometry to probe effects of ligand binding on G protein-coupled receptors.

Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

BACKGROUND: All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. METHODS: Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. RESULTS: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. CONCLUSION: Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

The Molecular Mechanism of P2Y1 Receptor Activation.

Human purinergic G protein-coupled receptor P2Y1 (P2Y1 R) is activated by adenosine 5'-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1 R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1 R activation.

Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells.

BACKGROUND/AIMS: Adenosine diphosphate ribose (ADPR), a product of ß-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. METHODS: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. RESULTS: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. CONCLUSION: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

Functional characterization of P2Y1 and P2X4 receptors in human neuroblastoma SK-N-MC cells / Caracterización funcional de receptores P2Y1 y P2X4 en células SK-N-MC de neuroblastoma humano

Nucleotides are important signalling molecules in both the peripheral and central nervous system. However, the in vitro study of their receptors can be hampered by the heterogeneity of primary neuronal cultures. The use of clonal neuroblastoma cell lines allows to circumvent this difficulty, so these lines are often used as a model to analyze the properties, regulation and physiological role of nucleotide receptors in neural tissues. Expression studies indicated the presence of P2Y1, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, P2X6 and P2X7 proteins in SK-N-MC cells. Functional analyses showed transient [Ca2+]i increases upon application of ADP, 2-MeSADP or ADPβS. Responses to these agonists seem to be mediated by a P2Y1 receptor, as demonstrated by the almost complete blockade exerted by the P2Y1-selective antagonist MRS2179. ATP was also able to induce [Ca2+]i increases in SK-N-MC cells. Responses to ATP were partially blocked by MRS2179 and the P2X antagonist TNP-ATP, thus suggesting that ATP can interact with two different P2 receptors: a P2Y1 receptor, inhibited by MRS2179, and a TNP-ATP sensitive P2X receptor. To characterize the P2X receptor responsible for the MRS2179-resistant component of the ATP response, we analyze the effect of several P2X agonists on [Ca2+]i. Cells did not show responses to either α,β-meATP or BzATP, although [Ca2+]i increases could be observed when cells were challenged with CTP. Both the response to CTP and the MRS2179-resistant component of ATP response were potentiated by ivermectin. Such pharmacological profile is consistent with the presence of a functional P2X4 receptor in SK-N-MC cell line

Los nucleótidos son importantes moléculas señalizadoras en el sistema nervioso. El estudio in vitro de sus receptores puede verse obstaculizado por la heterogeneidad de los cultivos neuronales. El uso de líneas celulares de neuroblastoma permite eludir esta dificultad y dichas líneas se utilizan frecuentemente como un modelo con el que analizar las propiedades, regulación y función de los receptores de nucleótidos en tejidos neurales. Estudios de expresión indicaron la presencia de proteínas P2Y1, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, P2X6 y P2X7 en las células SK-N-MC. Análisis funcionales mostraron incrementos transitorios de [Ca2+]i tras la aplicación de ADP, 2- MeSADP o ADPβS, respuestas que parecen estar mediadas a través un receptor P2Y1, como se pone de manifiesto por el bloqueo casi total ejercido por el antagonista selectivo P2Y1, MRS2179. El ATP también indujo incrementos de [Ca2+]i en las células SK-N-MC, siendo su respuesta parcialmente bloqueada por MRS2179 y por el antagonista P2X TNP-ATP, lo que sugiere que el ATP puede interactuar con dos receptores P2 diferentes: un receptor P2Y1, inhibido por MRS2179, y un receptor P2X sensible a TNP-ATP. Se caracterizó el receptor P2X analizando el efecto de varios agonistas en la [Ca2+]i. Ninguna célula mostró respuestas a α,β- meATP o BzATP, aunque se observaron incrementos de [Ca2+]i cuando las células fueron estimuladas con CTP. Tanto la respuesta a CTP como el componente de la respuesta a ATP resistente a MRS2179, se potenciaron en presencia de ivermectina. Todos estos datos sugieren la presencia de un receptor P2X4 funcional en las células SK-N-MC

Two disparate ligand-binding sites in the human P2Y1 receptor.

In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

Analyzing receptor assemblies in the cell membrane using fluorescence anisotropy imaging with TIRF microscopy.

Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.

Identification of 1-{2-[4-chloro-1'-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4'-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y(1) antagonist as an antiplatelet agent.

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.

Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor.

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models. By using small interfering RNA knockdowns, pharmacologic inhibitors and extracellular domains of the receptors TLR2, TLR4, RAGE, and P2Y1 as competitive inhibitors, we demonstrate that polyP amplifies H4- and HMGB1-mediated inflammatory signaling in human umbilical vein endothelial cells specifically through interaction with the RAGE and P2Y1 receptors, thereby eliciting intracellular Ca(2+) release. Finally, we demonstrate that the natural anticoagulant protease, activated protein C, potently inhibits polyP-mediated proinflammatory effects of both nuclear proteins in cellular and in vivo systems.

Potent P2Y1 urea antagonists bearing various cyclic amine scaffolds.

A number of new amine scaffolds with good inhibitory activity in the ADP-induced platelet aggregation assay have been found to be potent antagonists of the P2Y1 receptor. SAR optimization led to the identification of isoindoline 3c and piperidine 4a which showed good in vitro binding and functional activities, as well as improved aqueous solubility. Among them, the piperidine 4a showed the best overall profile with favorable PK parameters.