RESUMEN
Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-beta activation that was mediated by proteinase-activated receptor-1 (PAR1) and integrin alphavbeta5. PAR1, alphavbeta5, and alpha-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.
Asunto(s)
Factor Xa/metabolismo , Lesión Pulmonar/metabolismo , Fibrosis Pulmonar/metabolismo , Actinas/metabolismo , Adulto , Anciano , Animales , Secuencia de Bases , Bleomicina/toxicidad , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Factor Xa/genética , Inhibidores del Factor Xa , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/sangre , Fibrosis Pulmonar Idiopática/etiología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Lesión Pulmonar/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Modelos Biológicos , Fibrosis Pulmonar/sangre , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor PAR-1/metabolismo , Receptores de Vitronectina/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.
Asunto(s)
Coagulación Sanguínea/fisiología , Células Dendríticas/metabolismo , Inflamación/metabolismo , Receptor PAR-1/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Inflamación/inmunología , Sistema Linfático/inmunología , Ratones , Ratones Endogámicos C57BL , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor Cross-Talk/fisiología , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Sepsis/inmunología , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. RESULTS: Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli. CONCLUSION: The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.
Asunto(s)
Cistitis/genética , Cistitis/metabolismo , Regulación de la Expresión Génica , Receptor PAR-1/metabolismo , Animales , Antígenos/inmunología , Calcio/metabolismo , Cromatina/metabolismo , Cistitis/inducido químicamente , Cistitis/inmunología , Femenino , Expresión Génica , Genoma , Inmunoprecipitación , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfolipasas A/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Receptor PAR-1/deficiencia , Fracciones Subcelulares/metabolismo , Sustancia P , Vejiga Urinaria/metabolismoRESUMEN
BACKGROUND: In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PAR)s. Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis. RESULTS: Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency. CONCLUSION: Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.
Asunto(s)
Cistitis/metabolismo , Receptor PAR-1/metabolismo , Animales , Antígenos/inmunología , Línea Celular , Cistitis/inducido químicamente , Cistitis/inmunología , Cistitis/patología , Edema/inducido químicamente , Granulocitos/patología , Humanos , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor PAR-1/agonistas , Receptor PAR-1/deficiencia , Receptor PAR-2/efectos de los fármacos , Receptor PAR-2/metabolismo , Receptores Proteinasa-Activados/metabolismo , Sustancia P , Vejiga Urinaria/metabolismo , Urotelio/citología , Urotelio/metabolismoRESUMEN
Novel bis(indolyl)maleimide pyridinophanes 3, 9a, 9b, 10a, 10b, and 11 were prepared by cobalt-mediated [2+2+2] cycloaddition of an appropriate alpha,omega-diyne with an N,N-dialkylcyanamide. These macrocyclic heterophanes were found to be potent, selective inhibitors of glycogen synthase kinase-3beta. An X-ray structure of a co-crystal of GSK-3beta and 3 (IC(50)=3nM) depicts the hydrogen bonding and hydrophobic interactions in the ATP-binding pocket of this serine/threonine protein kinase.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Piridinas/farmacología , Adenosina Trifosfato/metabolismo , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Interacciones Hidrofóbicas e Hidrofílicas , Maleimidas/química , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Piridinas/química , Relación Estructura-ActividadRESUMEN
The transactivation of enhanced growth factor receptor (EGFR) by G protein-coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease-dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., "criss-cross" transactivation.
Asunto(s)
Receptores ErbB/metabolismo , Estrógenos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Línea Celular Tumoral , Receptores ErbB/efectos de los fármacos , Femenino , Humanos , Modelos Biológicos , Activación TranscripcionalRESUMEN
BACKGROUND: Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. METHODS AND RESULTS: Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. CONCLUSIONS: We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.
Asunto(s)
Plaquetas , Fragmentos de Péptidos/farmacología , Receptor PAR-1/antagonistas & inhibidores , Receptores de Trombina/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Animales , Línea Celular , Quimiotaxis , Dimerización , Modelos Animales de Enfermedad , Quimioterapia Combinada , Cobayas , Hirudinas/farmacología , Humanos , Fragmentos de Péptidos/uso terapéutico , Agregación Plaquetaria , Unión Proteica , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Trombina/metabolismo , Trombosis/etiología , TransfecciónRESUMEN
Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Perfilación de la Expresión Génica/métodos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Industria Farmacéutica/métodos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Análisis por Matrices de Proteínas/métodos , Proteína Quinasa C/análisis , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K(i) = 38 nm) and mast cell chymase (K(i) = 2.3 nm). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 A) and human chymase (1.90 A) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S(1) and S(2) subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S(1), the 1-naphthyl in S(2), and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S(3)/S(4) subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1alpha, interleukin-1beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Leucocitos/enzimología , Organofosfonatos/farmacología , Piperidinas/farmacología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Enfermedad Aguda , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/química , Catepsina G , Quimasas , Cristalografía por Rayos X , Humanos , Leucocitos/efectos de los fármacos , Masculino , Mastocitos/enzimología , Organofosfonatos/administración & dosificación , Organofosfonatos/química , Peritonitis/tratamiento farmacológico , Peritonitis/enzimología , Piperidinas/química , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Ratas , Ratas Sprague-Dawley , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/químicaRESUMEN
Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.
Asunto(s)
Indazoles/síntesis química , Indoles/síntesis química , Maleimidas/síntesis química , Proteína Quinasa C/antagonistas & inhibidores , Animales , Línea Celular , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Cobayas , Humanos , Indazoles/farmacología , Indazoles/toxicidad , Indoles/farmacología , Indoles/toxicidad , Interleucina-8/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Síndrome de QT Prolongado/inducido químicamente , Maleimidas/farmacología , Maleimidas/toxicidad , Modelos Moleculares , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , Proteína Quinasa C/química , Proteína Quinasa C beta , Relación Estructura-ActividadRESUMEN
The biopharmaceutical industry is currently being presented with opportunities to improve research and business efficiency via automation and the integration of various systems. In the examples discussed, industrial high-throughput screening systems are integrated with functional tools and bioinformatics to facilitate target and biomarker identification and validation. These integrative functional approaches generate value-added opportunities by leveraging available automation and information technologies into new applications that are broadly applicable to different types of projects, and by improving the overall research and development and business efficiency via the integration of various systems.
Asunto(s)
Automatización , Biología Computacional , Informática , Genómica/métodos , Genómica/tendencias , Humanos , Tecnología Farmacéutica/métodosRESUMEN
Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.
Asunto(s)
Proteínas Bacterianas/química , Biotina/química , Membranas Artificiales , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Esfingosina/química , Estreptavidina/química , Humanos , Cinética , Especificidad por SustratoRESUMEN
The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.
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Evaluación Preclínica de Medicamentos/métodos , ARN Interferente Pequeño/genética , Secuencia de Bases , Línea Celular , Biología Computacional , Evaluación Preclínica de Medicamentos/instrumentación , Genómica , Humanos , Reacción en Cadena de la Polimerasa/métodos , Robótica , TransfecciónRESUMEN
OBJECTIVE: Recently, mice made deficient in growth arrest-specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. METHODS AND RESULTS: Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine-induced pulmonary thromoembolism and inhibited ferric chloride-induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. CONCLUSIONS: Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.
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Plaquetas/enzimología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Pruebas de Coagulación Sanguínea , Plaquetas/fisiología , Western Blotting , Cloruros , Colágeno/farmacología , Colágeno/toxicidad , Epinefrina/toxicidad , Femenino , Compuestos Férricos/toxicidad , Humanos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligopéptidos/farmacología , Proteínas Oncogénicas/análisis , Agregación Plaquetaria/efectos de los fármacos , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Trombina/agonistas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tromboembolia/inducido químicamente , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Inhibidores de Proteínas Quinasas/química , Línea Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Maleimidas/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.
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Fibrinolisina/fisiología , Activación Plaquetaria , Receptores de Trombina/metabolismo , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Fibrinolisina/metabolismo , Humanos , Cinética , Ratones , Oligopéptidos/farmacología , Receptor PAR-1/metabolismo , Receptores de Trombina/agonistas , Receptores de Trombina/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Trombina/farmacologíaRESUMEN
Protease activated receptor-1 (PAR-1) is a key mediator of the cellular actions of alpha-thrombin. Thus, antagonism of this unique G-protein coupled receptor with a small molecule represents a means of selectively inhibiting thrombin's cellular actions without inhibiting its proteolytic activity. RWJ-58259 (alphaS)-N-[(1S)-3-amino-1-[[(phenylmethyl)- amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide) is a potent and selective inhibitor of PAR-1 identified as part of a synthetic chemistry program based upon a de novo design approach. RWJ-58259 inhibited thrombin-induced platelet aggregation in human platelets with an IC50 of 0.37 microM without inhibiting thrombin's proteolytic activity or aggregation induced by other agonists. RWJ-58259 was not effective in guinea pig models of thrombosis. This reflected the presence of a second thrombin-sensitive receptor system in guinea pigs (PAR-3/4) and the selectivity of RWJ-58259 for PAR-1. However, RWJ-58259 was effective in a non-human primate model of thrombosis. Because human platelets have a PAR expression profile similar to the non-human primate, PAR-1 antagonism has the potential to be antithrombotic in humans. RWJ-58259 also inhibited thrombin-induced intracellular calcium signaling and proliferation in rat vascular smooth muscle cells. Perivascular application of RWJ-58259 in vivo significantly inhibited arterial injury-induced stenosis in a rat model of balloon angioplasty. These preclinical results suggest a potential clinical utility of RWJ-58259 for treatment of thrombotic disorders and vascular injury associated with acute coronary interventions and atherosclerosis. Given the potential role of PAR-1 in thrombin's actions in other cell types and disease states, RWJ-58259 provides a means for assessing additional clinical utilities of PAR-1 antagonism in disease conditions such as inflammation, cancer and neurodegeneration.
Asunto(s)
Indazoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Receptor PAR-1/antagonistas & inhibidores , Urea/análogos & derivados , Urea/farmacología , Animales , Disponibilidad Biológica , Semivida , Humanos , Indazoles/química , Indazoles/farmacocinética , Músculo Liso Vascular/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Urea/química , Urea/farmacocinéticaRESUMEN
Efficient methods were developed to synthesize a novel series of macrocyclic bisindolylmaleimides containing linkers with multiple heteroatoms. Potent inhibitors (single digit nanomolar IC(50)) for PKC-beta and GSK-3beta were identified, and compounds showed good selectivity over PKC-alpha, -gamma, -delta, -epsilon, and -zeta. Representative compound 5a also had high selectivity in a screening panel of 10 other protein kinases. In cell-based functional assays, several compounds effectively blocked interleukin-8 release induced by PKC-betaII and increased glycogen synthase activity by inhibiting GSK-3beta.
Asunto(s)
Indoles/síntesis química , Maleimidas/síntesis química , Proteína Quinasa C/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular , Ciclización , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Humanos , Indoles/farmacología , Concentración 50 Inhibidora , Isoenzimas/síntesis química , Isoenzimas/farmacología , Maleimidas/farmacología , Proteína Quinasa C beta , Relación Estructura-ActividadRESUMEN
A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor (PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC(50)=25 and 35 nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH(2) (TRAP-6) and alpha-thrombin.
Asunto(s)
Indoles/síntesis química , Indoles/farmacología , Receptores de Trombina/efectos de los fármacos , Urea/análogos & derivados , Aminas/química , Hemostáticos/antagonistas & inhibidores , Hemostáticos/farmacología , Humanos , Técnicas In Vitro , Indazoles/química , Ligandos , Imitación Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Relación Estructura-Actividad , Trombina/antagonistas & inhibidores , Trombina/farmacología , Urea/químicaRESUMEN
It is well appreciated that thrombin as well as other proteases can act as signaling molecules that specifically regulate cells by cleaving and activating members of a novel class of protease-activated receptors (PARs). The utility of gene knockout strategies to define and better comprehend the physiological role of specific proteins is perhaps best exemplified in the field of thrombin receptors. The development of PAR knockout mice has provided the unique opportunity to identify and characterize new members of this novel family of GPCRs, evaluate the interaction of PARs jointly expressed in common cells and tissues, and better understand the role of PARs in thrombosis, restenosis, vascular remodeling, angiogenesis, and inflammation. Presently, 4 members of the PAR family have been cloned and identified. In this review, we examine experimental evidence gleaned from PAR-/- mouse models as well as how the use of PAR-/- mice has provided insights toward understanding the physiological role of thrombin in cells of the vascular system and vascular pathology.