Browse
Submit Data
Databases
API
Help

Dataset Information

6 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Synthesis of indole derived protease-activated receptor 4 antagonists and characterization in human platelets.

ABSTRACT: Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development.

SUBMITTER: Young SE

PROVIDER: S-EPMC3679140 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Synthesis of indole derived protease-activated receptor 4 antagonists and characterization in human platelets.

Young Summer E SE Duvernay Matthew T MT Schulte Michael L ML Lindsley Craig W CW Hamm Heidi E HE

PloS one 20130611 6

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subse ...[more]

PMID: 23776495

Similar Datasets

Cloning and characterization of human protease-activated receptor 4.

Project description:Protease-activated receptors 1-3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47/Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By fluorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

| S-EPMC22580 | biostudies-literature

Protease-activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.

Project description:With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

| S-EPMC3608438 | biostudies-literature

Potent inhibition of breast cancer by bis-indole-derived nuclear receptor 4A1 (NR4A1) antagonists.

Project description:BACKGROUND:Nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors, and the methylene-substituted bis-indole derivative 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) acts as an NR4A1 antagonist (inverse agonist) and inhibits NR4A1-regulated pro-oncogenic pathways/genes in breast and other cancer cells. METHODS:Buttressed analogs of DIM-C-pPhOH were synthesized by condensation of the substituted p-hydroxybenzaldehydes with indole. Breast cancer cell growth, survival, and migration assays were carried out by cell counting, Annexin V staining, and Boyden chamber assays, respectively. Changes in RNA and protein expression were determined by RT-PCR and western blots, respectively. Analysis of RNAseq results was carried out using Ingenuity Pathway Analysis, and in vivo potencies of NR4A1 antagonists were determined in athymic nude mice bearing MDA-MB-231 cells in an orthotopic model. RESULTS:Ingenuity Pathway analysis of common genes modulated by NR4A1 knockdown or treatment with DIM-C-pPhOH showed that changes in gene expression were consistent with the observed decreased functional responses, namely inhibition of growth and migration and increased apoptosis. DIM-C-pPhOH is rapidly metabolized and the effects and potencies of buttressed analogs of DIM-C-pPhOH which contain one or two substituents ortho to the hydroxyl groups were investigated using NR4A1-regulated gene/gene products as endpoints. The buttressed analogs were more potent than DIM-C-pPhOH in both in vitro assays and as inhibitors of mammary tumor growth. Moreover, using 1,1-bis(3'-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (DIM-C-pPhOh-3-Cl-5-OCH3) significant tumor growth inhibition was observed at doses as low as 2 mg/kg/d which was at least an order of magnitude more potent than DIM-C-pPhOH. CONCLUSIONS:These buttressed analogs represent a more potent set of second generation NR4A1 antagonists as inhibitors of breast cancer.

| S-EPMC6681651 | biostudies-literature

Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin.

Project description:Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents.

| S-EPMC408153 | biostudies-literature

Synthesis and biological characterization of protease-activated prodrugs of doxazolidine.

Project description:Doxazolidine (doxaz) is a new anthracycline anticancer agent. While structurally similar to doxorubicin (dox), doxaz acts via a distinct mechanism to selectively enhance anticancer activity over cardiotoxicity, the most significant clinical impediment to successful anthracycline treatment. Here, we describe the synthesis and characterization of a prodrug platform designed for doxaz release mediated by secreted proteolytic activity, a common association with invasiveness and poor prognosis in cancer patients. GaFK-Doxaz is hydrolyzable by the proteases plasmin and cathepsin B, both strongly linked with cancer progression, as well as trypsin. We demonstrate that activation of GaFK-Doxaz releases highly potent doxaz that powerfully inhibits the growth of a wide variety of cancer cells (average IC(50) of 8 nM). GaFK-Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.

| S-EPMC3433255 | biostudies-literature

Bis-indole derived nuclear receptor 4A1 (NR4A1) antagonists inhibit TGFβ-induced invasion of embryonal rhabdomyosarcoma cells.

Project description:Transforming growth factor β (TGFβ) enhances invasion of breast and lung cancer cells through phosphorylation-dependent nuclear export of the nuclear receptor 4A1 (NR4A1, Nur77). This response is inhibited by the NR4A1 antagonist 1,1-bis(3'-indoly)-1-(p-hydroxyphenyl) methane (CDIM8) and we hypothesized that similar effects would be observed in Rhabdomyosarcoma (RMS) cells. Although some kinase inhibitors block TGFβ-induced invasion of embryonal RMS (ERMS) cells, the mechanism differs from breast and lung cancer cells since NR4A1 is extranuclear in ERMS cells. However, CDIM8 blocks basal and TGFβ-induced invasion of RD and SMS-CTR ERMS cell lines but not Rh30 alveolar RMS (ARMS) cells. Moreover, this response in ERMS cells was independent of SMAD7 degradation or activation of SMAD2/SMAD3. β-Catenin silencing decreased ERMS cell invasion and CDIM8 induced proteasome-independent downregulation of β-catenin. The novel mechanism of CDIM8-mediated inhibition of basal and TGFβ-induced ERMS cell invasion was due to activation of the Bcl-2-NR4A1 complex, mitochondrial disruption, induction of the tumor suppressor-like cytokine interleukin-24 (IL-24) which in turn downregulates β-catenin expression. Thus, the NR4A1 antagonist inhibits TGFβ-induced invasion of ERMS cells through initial targeting of cytosolic NR4A1.

| S-EPMC7471359 | biostudies-literature

Protease-activated receptor-2 modulates protease-activated receptor-1-driven neointimal hyperplasia.

Project description:Emerging evidence suggests that protease-activated receptors-1 and -2 (PAR1 and PAR2) can signal together in response to proteases found in the rapidly changing microenvironment of damaged blood vessels. However, it is unknown whether PAR1 and PAR2 promote or mitigate the hyperplastic response to arterial injury. Using cell-penetrating PAR1 pepducins and mice deficient in PAR1 or PAR2, we set out to determine the respective contributions of the receptors to hyperplasia and phenotypic modulation of smooth muscle cells (SMCs) in response to arterial injury.SMCs were strongly activated by PAR1 stimulation, as evidenced by increased mitogenesis, mitochondrial activity, and calcium mobilization. The effects of chronic PAR1 stimulation following vascular injury were studied by performing carotid artery ligations in mice treated with the PAR1 agonist pepducin, P1pal-13. Histological analysis revealed that PAR1 stimulation caused striking hyperplasia, which was ablated in PAR1(-/-) and, surprisingly, PAR2(-/-) mice. P1pal-13 treatment yielded an expression pattern consistent with a dedifferentiated phenotype in carotid artery SMCs. Detection of PAR1-PAR2 complexes provided an explanation for the hyperplastic effects of the PAR1 agonist requiring the presence of both receptors.We conclude that PAR2 regulates the PAR1 hyperplastic response to arterial injury leading to stenosis.

| S-EPMC3241440 | biostudies-literature

Synthesis, SAR, and Pharmacological Characterization of Brain Penetrant P2X7 Receptor Antagonists.

Project description:We describe the synthesis and SAR of 1,2,3-triazolopiperidines as a novel series of potent, brain penetrant P2X7 antagonists. Initial efforts yielded a series of potent human P2X7R antagonists with moderate to weak rodent potency, some CYP inhibition, poor metabolic stability, and low solubility. Further work in this series, which focused on the SAR of the N-linked heterocycle, not only increased the potency at the human P2X7R but also provided compounds with good potency at the rat P2X7R. These efforts eventually delivered a potent rat and human P2X7R antagonist with good physicochemical properties, an excellent pharmacokinetic profile, good partitioning into the CNS, and demonstrated in vivo target engagement after oral dosing.

| S-EPMC4468405 | biostudies-literature

Protease-activated receptor signaling in platelets activates cytosolic phospholipase A2? differently for cyclooxygenase-1 and 12-lipoxygenase catalysis.

Project description:The rate-limiting step in the biosynthesis of thromboxane A(2) (TxA(2)) and 12-hydroxyeicosatetraenoic acid (12-HETE) by platelets is activation of cytosolic phospholipase A(2?) (cPLA(2?)), which releases arachidonic acid, which is the substrate for cyclooxygenase-1 (COX-1) and 12-lipoxygenase. We evaluated signaling via the human platelet thrombin receptors, protease-activated receptor (PAR) 1 and PAR4, to the activation of cPLA(2?), which provides a substrate for the biosynthesis of TxA(2) and 12-HETE.Stimulating washed human platelets resulted in delayed biosynthesis of 12-HETE, which continues after maximal formation of TxA(2) is completed, suggesting that 12-HETE is not formed by the same pool of arachidonic acid that provides a substrate to COX-1. PAR1-induced formation of TxA(2) was inhibited by the phosphatidylinositol kinase inhibitor LY294002, whereas this inhibitor did not block 12-HETE biosynthesis. Both 1-butanol and propranolol also blocked TxA(2) biosynthesis but did not inhibit 12-HETE formation.The concerted evidence indicates that the platelet thrombin receptors signal activation of cPLA(2?) coupled to COX-1 by a pathway different from that signaling activation of the cPLA(2?) coupled to 12-lipoxygenase.

| S-EPMC3035574 | biostudies-literature

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα.

Project description:BackgroundPlatelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated.ObjectiveTo investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation.Methods and resultsHighly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins.ConclusionsBoth GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.

| S-EPMC4656914 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data