RESUMEN
Flow photolysis of aryldiazoacetates 3-5 leads to C-H insertion to form dihydrobenzofurans 6-8 in a metal-free process, using either a medium pressure mercury lamp (250-390 nm) or LEDs (365 nm or 450 nm) with comparable synthetic outcomes. Significantly, addition of 4,4'-dimethoxybenzophenone 9 results in an increased yield and also alters the stereochemical outcome leading to preferential isolation of the trans dihydrobenzofurans 6a-8a (up to 50% yield), while the cis and trans diastereomers of 6-8 are recovered in essentially equimolar amounts in the absence of a photosensitiser (up to 26% yield).
RESUMEN
Generation of tosyl azide 12 in acetonitrile in flow under water-free conditions using an azide resin and its use in diazo transfer to a series of aryl acetates are described. Successful telescoping with a rhodium acetate-catalyzed O-H insertion has been achieved, thereby transforming the aryl acetate 8 to α-hydroxy ester 10, a key intermediate in the synthesis of clopidogrel 11, without requiring isolation or handling of either tosyl azide 12 or α-aryl-α-diazoacetate 9, or indeed having significant amounts of either present at any point. Significantly, the solution of α-diazo ester 9 was sufficiently clean to progress directly to the rhodium acetate-catalyzed step without any detrimental impact on the efficiency of the O-H insertion. In addition, the rhodium acetate-catalyzed O-H insertion process is cleaner in flow than under traditional batch conditions. Use of the azide resin offers clear safety advantages and, in addition, this approach complements earlier protocols for the generation of tosyl azide 12 in flow; this protocol is especially useful with less acidic substrates.
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HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.
Asunto(s)
Benzoquinonas/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Taxoides/administración & dosificación , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Benzoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Regulación hacia Abajo , Sinergismo Farmacológico , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/metabolismo , Masculino , Taxoides/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Thrombin-dependent platelet activation is heightened in the setting of percutaneous coronary intervention and may cause arterial thrombosis with consequent myocardial necrosis. Given the high incidence of adverse effects in patients with acute coronary syndromes, there remains an unmet need for the development of new therapeutics that target platelet activation without unduly affecting hemostasis. The thrombin receptor, PAR1, has recently emerged as a promising new target for therapeutic intervention in patients with acute coronary syndromes. METHODS AND RESULTS: We report the development of a first-in-class intracellular PAR1 inhibitor with optimized pharmacokinetic properties for use during percutaneous coronary intervention in patients with acute coronary syndromes. PZ-128 is a cell-penetrating pepducin inhibitor of PAR1 that targets the receptor-G-protein interface on the inside surface of platelets. The structure of PZ-128 closely resembles the predicted off-state of the corresponding juxtamembrane region of the third intracellular loop of PAR1. The onset of action of PZ-128 was rapid and suppressed PAR1 aggregation and arterial thrombosis in guinea pigs and baboons and strongly synergized with oral clopidogrel. There was full recovery of platelet function by 24 hours. Importantly, PZ-128 had no effect on bleeding or coagulation parameters in primates or in blood from patients undergoing percutaneous coronary intervention. CONCLUSIONS: Based on the efficacy data in nonhuman primates with no noted adverse effects on hemostasis, we anticipate that the rapid onset of platelet inhibition and reversible properties of PZ-128 are well suited to the acute interventional setting of percutaneous coronary intervention and may provide an alternative to long-acting small-molecule inhibitors of PAR1.
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Membrana Celular/efectos de los fármacos , Hemostasis/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Receptor PAR-1/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Animales , Membrana Celular/metabolismo , Cobayas , Hemostasis/fisiología , Humanos , Lipopéptidos/uso terapéutico , Macaca fascicularis , Masculino , Papio anubis , Activación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Receptor PAR-1/metabolismo , Trombosis/metabolismoRESUMEN
Matrix metalloprotease-1 (MMP1), a collagenase and activator of the G protein-coupled protease activated receptor-1 (PAR1), is an emerging new target implicated in oncogenesis and metastasis in diverse cancers. However, the functional mouse homologue of MMP1 in cancer models has not yet been clearly defined. We report here that Mmp1a is a functional MMP1 homologue that promotes invasion and metastatic progression of mouse lung cancer and melanoma. LLC1 (Lewis lung carcinoma) and primary mouse melanoma cells harboring active BRAF express high levels of endogenous Mmp1a, which is required for invasion through collagen. Silencing of either Mmp1a or PAR1 suppressed invasive stellate growth of lung cancer cells in three-dimensional matrices. Conversely, ectopic expression of Mmp1a conferred an invasive phenotype in epithelial cells that do not express endogenous Mmp1a. Consistent with Mmp1a acting as a PAR1 agonist in an autocrine loop, inhibition or silencing of PAR1 resulted in a loss of the Mmp1a-driven invasive phenotype. Knockdown of Mmp1a on tumor cells resulted in significantly decreased tumorigenesis, invasion, and metastasis in xenograft models. Together, these data demonstrate that cancer cell-derived Mmp1a acts as a robust functional homologue of MMP1 by conferring protumorigenic and metastatic behavior to cells.
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Metaloproteinasa 1 de la Matriz/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Metaloproteinasa 1 de la Matriz/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor PAR-1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gα(q/11/o/13)), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets.
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Diseño de Fármacos , Péptidos/química , Péptidos/farmacología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.
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Leucemia Linfoide/tratamiento farmacológico , Lipopéptidos/uso terapéutico , Linfoma/tratamiento farmacológico , Receptores CXCR4/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patología , Lipopéptidos/administración & dosificación , Lipopéptidos/síntesis química , Lipopéptidos/química , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Modelos Moleculares , Terapia Molecular Dirigida , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer.
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Adenocarcinoma/tratamiento farmacológico , Lipopéptidos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Apoptosis , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Citoplasma/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Lipopéptidos/farmacocinética , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptor PAR-1/genética , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Distribución TisularRESUMEN
Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H(+)-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.
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Endosomas/efectos de los fármacos , Endosomas/enzimología , Hemoproteínas/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Receptores de Transferrina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hemoproteínas/genética , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ratones , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Saccharomyces cerevisiae/metabolismoRESUMEN
Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor that is not expressed in normal breast epithelia but is up-regulated in invasive breast carcinomas. In the present study, we found that matrix metalloprotease-1 (MMP-1) robustly activates the PAR1-Akt survival pathway in breast carcinoma cells. This process is blocked by a cell-penetrating lipopeptide "pepducin," P1pal-7, which is a potent inhibitor of cell viability in breast carcinoma cells expressing PAR1. Both a MMP-1 inhibitor and P1pal-7 significantly promote apoptosis in breast tumor xenografts and inhibit metastasis to the lungs by up to 88%. Dual therapy with P1pal-7 and Taxotere inhibits the growth of MDA-MB-231 xenografts by 95%. Consistently, biochemical analysis of xenograft tumors treated with P1pal-7 or MMP-1 inhibitor showed attenuated Akt activity. Ectopic expression of constitutively active Akt rescues breast cancer cells from the synergistic cytotoxicity of P1pal-7 and Taxotere, suggesting that Akt is a critical component of PAR1-dependent cancer cell viability. Together, these findings indicate that blockade of MMP1-PAR1 signaling may provide a benefit beyond treatment with Taxotere alone in advanced, metastatic breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Receptor PAR-1/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Docetaxel , Sinergismo Farmacológico , Activación Enzimática , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Metaloproteinasa 1 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Desnudos , Péptidos/administración & dosificación , Péptidos/farmacocinética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Transducción de Señal/efectos de los fármacos , Taxoides/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.
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Receptor PAR-1/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/metabolismo , Colágeno/metabolismo , Proteínas de Unión al GTP/metabolismo , Cobayas , Humanos , Ligandos , Metaloproteinasa 1 de la Matriz/metabolismo , Estructura Terciaria de Proteína , Receptor PAR-1/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The insulin/insulin-like growth factor (IGF) signaling pathway to mTOR is essential for the survival and growth of normal cells and also contributes to the genesis and progression of cancer. This signaling pathway is linked with regulation of mitochondrial function, but how is incompletely understood. Here we show that IGF-I and insulin induce rapid transcription of the mitochondrial pyrimidine nucleotide carrier PNC1, which shares significant identity with the essential yeast mitochondrial carrier Rim2p. PNC1 expression is dependent on PI-3 kinase and mTOR activity and is higher in transformed fibroblasts, cancer cell lines, and primary prostate cancers than in normal tissues. Overexpression of PNC1 enhances cell size, whereas suppression of PNC1 expression causes reduced cell size and retarded cell cycle progression and proliferation. Cells with reduced PNC1 expression have reduced mitochondrial UTP levels, but while mitochondrial membrane potential and cellular ATP are not altered, cellular ROS levels are increased. Overall the data indicate that PNC1 is a target of the IGF-I/mTOR pathway that is essential for mitochondrial activity in regulating cell growth and proliferation.