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1.
Cell ; 137(2): 332-43, 2009 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-19379698

RESUMEN

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.


Asunto(s)
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/metabolismo
2.
Hepatology ; 76(6): 1778-1793, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-35603482

RESUMEN

BACKGROUND AND AIMS: Insulin resistance and poor glycemic control are key drivers of the development of NAFLD and have recently been shown to be associated with fibrosis progression in NASH. However, the underlying mechanisms involving dysfunctional glucose metabolism and relationship with NAFLD/NASH progression remain poorly understood. We set out to determine whether protease-activated receptor 2 (PAR2), a sensor of extracellular inflammatory and coagulation proteases, links NAFLD and NASH with liver glucose metabolism. APPROACH AND RESULTS: Here, we demonstrate that hepatic expression of PAR2 increases in patients and mice with diabetes and NAFLD/NASH. Mechanistic studies using whole-body and liver-specific PAR2-knockout mice reveal that hepatic PAR2 plays an unexpected role in suppressing glucose internalization, glycogen storage, and insulin signaling through a bifurcating Gq -dependent mechanism. PAR2 activation downregulates the major glucose transporter of liver, GLUT2, through Gq -MAPK-FoxA3 and inhibits insulin-Akt signaling through Gq -calcium-CaMKK2 pathways. Therapeutic dosing with a liver-homing pepducin, PZ-235, blocked PAR2-Gq signaling and afforded significant improvements in glycemic indices and HbA1c levels in severely diabetic mice. CONCLUSIONS: This work provides evidence that PAR2 is a major regulator of liver glucose homeostasis and a potential target for the treatment of diabetes and NASH.


Asunto(s)
Diabetes Mellitus Experimental , Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptor PAR-2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Hígado/metabolismo , Insulina/metabolismo , Glucosa/metabolismo , Ratones Noqueados
3.
Arterioscler Thromb Vasc Biol ; 41(1): e33-e45, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33267659

RESUMEN

OBJECTIVE: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. CONCLUSIONS: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.


Asunto(s)
Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/sangre , Hemostasis , Agregación Plaquetaria , Receptores Acoplados a Proteínas G/sangre , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangre , Animales , Células CHO , Trombosis de las Arterias Carótidas/prevención & control , Cricetulus , Modelos Animales de Enfermedad , Femenino , Fibrinolíticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/sangre , Humanos , Masculino , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Trombina/sangre , Transducción de Señal , Trombina/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 41(5): e265-e279, 2021 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-33761760
5.
Arterioscler Thromb Vasc Biol ; 40(12): 2990-3003, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33028101

RESUMEN

OBJECTIVE: Arterial thrombosis leading to ischemic injury worsens the prognosis of many patients with cardiovascular disease. PZ-128 is a first-in-class pepducin that reversibly inhibits PAR1 (protease-activated receptor 1) on platelets and other vascular cells by targeting the intracellular surface of the receptor. The TRIP-PCI (Thrombin Receptor Inhibitory Pepducin in Percutaneous Coronary Intervention) trial was conducted to assess the safety and efficacy of PZ-128 in patients undergoing cardiac catheterization with intent to perform percutaneous coronary intervention. Approach and Results: In this randomized, double-blind, placebo-controlled, phase 2 trial, 100 patients were randomly assigned (2:1) to receive PZ-128 (0.3 or 0.5 mg/kg), or placebo in a 2-hour infusion initiated just before the start of cardiac catheterization, on top of standard oral antiplatelet therapy. Rates of the primary end point of bleeding were not different between the combined PZ-128 doses (1.6%, 1/62) and placebo group (0%, 0/35). The secondary end points of major adverse coronary events at 30 and 90 days did not significantly differ but were numerically lower in the PZ-128 groups (0% and 2% in the PZ-128 groups, 6% and 6% with placebo, p=0.13, p=0.29, respectively). In the subgroup of patients with elevated baseline cardiac troponin I, the exploratory end point of 30-day major adverse coronary events + myocardial injury showed 83% events in the placebo group versus 31% events in the combined PZ-128 drug groups, an adjusted relative risk of 0.14 (95% CI, 0.02-0.75); P=0.02. CONCLUSIONS: In this first-in-patient experience, PZ-128 added to standard antiplatelet therapy appeared to be safe, well tolerated, and potentially reduced periprocedural myonecrosis, thus providing the basis for further clinical trials. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02561000.


Asunto(s)
Síndrome Coronario Agudo/terapia , Plaquetas/efectos de los fármacos , Cateterismo Cardíaco , Péptidos de Penetración Celular/administración & dosificación , Enfermedad de la Arteria Coronaria/terapia , Lipopéptidos/administración & dosificación , Miocardio/patología , Intervención Coronaria Percutánea , Inhibidores de Agregación Plaquetaria/administración & dosificación , Receptor PAR-1/agonistas , Trombosis/prevención & control , Síndrome Coronario Agudo/diagnóstico por imagen , Anciano , Plaquetas/metabolismo , Cateterismo Cardíaco/efectos adversos , Cateterismo Cardíaco/instrumentación , Péptidos de Penetración Celular/efectos adversos , Péptidos de Penetración Celular/farmacocinética , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Método Doble Ciego , Femenino , Humanos , Infusiones Intravenosas , Lipopéptidos/efectos adversos , Lipopéptidos/farmacocinética , Masculino , Persona de Mediana Edad , Necrosis , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/instrumentación , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/farmacocinética , Prueba de Estudio Conceptual , Estudios Prospectivos , Receptor PAR-1/metabolismo , Recurrencia , Stents , Trombosis/sangre , Trombosis/etiología , Factores de Tiempo , Resultado del Tratamiento , Estados Unidos
6.
J Biol Chem ; 294(12): 4401-4411, 2019 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-30670596

RESUMEN

Pancreatic ß-cell failure in type 2 diabetes mellitus is a serious challenge that results in an inability of the pancreas to produce sufficient insulin to properly regulate blood glucose levels. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor expressed by ß-cells that has recently been proposed as a potential target for improving glycemic control and suppressing binge eating behaviors. We discovered that TAAR1 is coupled to Gαs-signaling pathways in insulin-secreting ß-cells to cause protein kinase A (PKA)/exchange protein activated by cAMP (Epac)-dependent release of insulin, activation of RAF proto-oncogene, Ser/Thr kinase (Raf)-mitogen-activated protein kinase (MAPK) signaling, induction of cAMP response element-binding protein (CREB)-insulin receptor substrate 2 (Irs-2), and increased ß-cell proliferation. Interestingly, TAAR1 triggered cAMP-mediated calcium influx and release from internal stores, both of which were required for activation of a MAPK cascade utilizing calmodulin-dependent protein kinase II (CaMKII), Raf, and MAPK/ERK kinase 1/2 (MEK1/2). Together, these data identify TAAR1/Gαs-mediated signaling pathways that promote insulin secretion, improved ß-cell function and proliferation, and highlight TAAR1 as a promising new target for improving ß-cell health in type 2 diabetes mellitus.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Adenilil Ciclasas/metabolismo , Animales , Calcio/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucosa/farmacología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Ratones , Fosforilación , Ratas , Receptores Acoplados a Proteínas G/agonistas
7.
Nat Immunol ; 8(12): 1303-12, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17965715

RESUMEN

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.


Asunto(s)
Células Endoteliales/fisiología , Receptor PAR-1/fisiología , Receptor PAR-2/fisiología , Sepsis/metabolismo , Transducción de Señal/fisiología , Animales , Permeabilidad Capilar , Comunicación Celular , Línea Celular , Ratones , Receptor PAR-1/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/metabolismo , Sepsis/fisiopatología , Enfermedades Vasculares/etiología
8.
Hepatology ; 77(5): E111-E112, 2023 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-36869845
9.
Arterioscler Thromb Vasc Biol ; 38(6): 1368-1380, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29622563

RESUMEN

OBJECTIVE: Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist. APPROACH AND RESULTS: We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E-deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1-PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells. CONCLUSIONS: These data suggest that targeting the MMP1-PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.


Asunto(s)
Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Enfermedades de las Arterias Carótidas/enzimología , Enfermedad de la Arteria Coronaria/enzimología , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Receptor PAR-1/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/patología , Aterosclerosis/prevención & control , Biomarcadores/sangre , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/prevención & control , Línea Celular , Péptidos de Penetración Celular/farmacología , Ensayos Clínicos Fase II como Asunto , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Fibrinolíticos/farmacología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Ácidos Hidroxámicos/farmacología , Lipopéptidos/farmacología , Masculino , Metaloproteinasa 1 de la Matriz/sangre , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Oligopéptidos/farmacología , Placa Aterosclerótica , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/sangre , Transducción de Señal , Factor de Necrosis Tumoral alfa/sangre , Estados Unidos
10.
Int J Mol Sci ; 19(8)2018 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-30065181

RESUMEN

The G-protein coupled receptors (GPCRs) belong to a large family of diverse receptors that are well recognized as pharmacological targets. However, very few of these receptors have been pursued as oncology drug targets. The Protease-activated receptor 1 (PAR1), which is a G-protein coupled receptor, has been shown to act as an oncogene and is an emerging anti-cancer drug target. In this paper, we provide an overview of PAR1's biased signaling role in metastatic cancers of the breast, lungs, and ovaries and describe the development of PAR1 inhibitors that are currently in clinical use to treat acute coronary syndromes. PAR1 inhibitor PZ-128 is in a Phase II clinical trial and is being developed to prevent ischemic and thrombotic complication of patients undergoing cardiac catheterization. PZ-128 belongs to a new class of cell-penetrating, membrane-tethered peptides named pepducins that are based on the intracellular loops of receptors targeting the receptor G-protein interface. Application of PZ-128 as an anti-metastatic and anti-angiogenic therapeutic agent in breast, lung, and ovarian cancer is being reviewed.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Receptor PAR-1/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Péptidos de Penetración Celular/uso terapéutico , Femenino , Humanos , Lipopéptidos/uso terapéutico , Neoplasias Pulmonares/metabolismo , Neoplasias Ováricas/metabolismo , Receptor PAR-1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos
11.
J Biol Chem ; 291(44): 23188-23198, 2016 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-27613872

RESUMEN

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.


Asunto(s)
Lipopéptidos/administración & dosificación , Cirrosis Hepática/prevención & control , Receptor PAR-2/metabolismo , Animales , Hepatocitos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Receptor PAR-2/antagonistas & inhibidores , Receptor PAR-2/genética
12.
Arterioscler Thromb Vasc Biol ; 36(1): 189-97, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26681756

RESUMEN

OBJECTIVE: Pepducins are membrane-tethered, cell-penetrating lipopeptides that target the cytoplasmic surface of their cognate receptor. Here, we report the first human use of a protease-activated receptor-1-based pepducin, which is intended as an antiplatelet agent to prevent ischemic complications of percutaneous coronary interventions. APPROACH AND RESULTS: PZ-128 was administered by 1 to 2 hours continuous intravenous infusion (0.01-2 mg/kg) to 31 subjects with coronary artery disease or multiple coronary artery disease risk factors. Safety, antiplatelet efficacy, and pharmacokinetics were assessed at baseline and 0.5, 1, 2, 6, 24 hours, and 7 to 10 days postdosing. The inhibitory effects of PZ-128 on platelet aggregation stimulated by the protease-activated receptor-1 agonist SFLLRN (8 µmol/L) at 30 minutes to 6 hours were dose dependent with 20% to 40% inhibition at 0.3 mg/kg, 40% to 60% at 0.5 mg/kg, and ≥ 80% to 100% at 1 to 2 mg/kg. The subgroup receiving aspirin in the 0.5 and 1-mg/kg dose cohorts had 65% to 100% inhibition of final aggregation to SFLLRN at 30 minutes to 2 hours and 95% to 100% inhibition by 6 hours. The inhibitory effects of 0.5 mg/kg PZ-128 were reversible with 50% recovery of aggregation to SFLLRN by 24 hours. There were no significant effects of PZ-128 on aggregation induced by AYPGKF, ADP, or collagen, indicating that the observed effects were specific to protease-activated receptor-1. The plasma half-life was 1.3 to 1.8 hours, and PZ-128 was nondetectable in urine. There were no effects on bleeding, coagulation, clinical chemistry, or ECG parameters. CONCLUSIONS: PZ-128 is a promising antiplatelet agent that provides rapid, specific, dose dependent, and reversible inhibition of platelet protease-activated receptor-1 through a novel intracellular mechanism. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01806077.


Asunto(s)
Plaquetas/efectos de los fármacos , Péptidos de Penetración Celular/administración & dosificación , Enfermedad de la Arteria Coronaria/terapia , Lipopéptidos/administración & dosificación , Intervención Coronaria Percutánea , Inhibidores de Agregación Plaquetaria/administración & dosificación , Agregación Plaquetaria/efectos de los fármacos , Receptor PAR-1/antagonistas & inhibidores , Adulto , Anciano , Plaquetas/metabolismo , Péptidos de Penetración Celular/efectos adversos , Péptidos de Penetración Celular/farmacocinética , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Humanos , Infusiones Intravenosas , Lipopéptidos/efectos adversos , Lipopéptidos/farmacocinética , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea/efectos adversos , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/farmacocinética , Pruebas de Función Plaquetaria , Receptor PAR-1/metabolismo , Resultado del Tratamiento
13.
J Biol Chem ; 290(25): 15785-15798, 2015 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-25934391

RESUMEN

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.


Asunto(s)
Materiales Biomiméticos/química , Péptidos de Penetración Celular/química , Subunidades alfa de la Proteína de Unión al GTP/química , Receptor PAR-1/química , Regulación Alostérica/genética , Secuencias de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Resonancia Magnética Nuclear Biomolecular , Receptor PAR-1/genética , Receptor PAR-1/metabolismo
14.
Blood ; 121(3): 431-9, 2013 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-23086754

RESUMEN

Cardiovascular diseases, including atherothrombosis, are the leading cause of morbidity and mortality in the United States, Europe, and the developed world. Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet and endothelial function, and atherothrombotic disease. Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that is classically activated through cleavage of the N-terminal exodomain by the serine protease thrombin. Most recently, 2 MMPs have been discovered to have agonist activity for PAR1. Unexpectedly, MMP-1 and MMP-13 cleave the N-terminal exodomain of PAR1 at noncanonical sites, which result in distinct tethered ligands that activate G-protein signaling pathways. PAR1 exhibits metalloprotease-specific signaling patterns, known as biased agonism, that produce distinct functional outputs by the cell. Here we contrast the mechanisms of canonical (thrombin) and noncanonical (MMP) PAR1 activation, the contribution of MMP-PAR1 signaling to diseases of the vasculature, and the therapeutic potential of inhibiting MMP-PAR1 signaling with MMP inhibitors, including atherothrombotic disease, in-stent restenosis, heart failure, and sepsis.


Asunto(s)
Aterosclerosis/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Receptor PAR-1/metabolismo , Transducción de Señal/fisiología , Trombosis/metabolismo , Animales , Humanos
15.
J Biol Chem ; 288(32): 23105-15, 2013 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-23814055

RESUMEN

Vascular injury that results in proliferation and dedifferentiation of vascular smooth muscle cells (SMCs) is an important contributor to restenosis following percutaneous coronary interventions or plaque rupture. Protease-activated receptor-1 (PAR1) has been shown to play a role in vascular repair processes; however, little is known regarding its function or the relative roles of the upstream proteases thrombin and matrix metalloprotease-1 (MMP-1) in triggering PAR1-mediated arterial restenosis. The goal of this study was to determine whether noncanonical MMP-1 signaling through PAR1 would contribute to aberrant vascular repair processes in models of arterial injury. A mouse carotid arterial wire injury model was used for studies of neointima hyperplasia and arterial stenosis. The mice were treated post-injury for 21 days with a small molecule inhibitor of MMP-1 or a direct thrombin inhibitor and compared with vehicle control. Intimal and medial hyperplasia was significantly inhibited by 2.8-fold after daily treatment with the small molecule MMP-1 inhibitor, an effect that was lost in PAR1-deficient mice. Conversely, chronic inhibition of thrombin showed no benefit in suppressing the development of arterial stenosis. Thrombin-PAR1 signaling resulted in a supercontractile, differentiated phenotype in SMCs. Noncanonical MMP-1-PAR1 signaling resulted in the opposite effect and led to a dedifferentiated phenotype via a different G protein pathway. MMP-1-PAR1 significantly stimulated hyperplasia and migration of SMCs, and resulted in down-regulation of SMC contractile genes. These studies provide a new mechanism for the development of vascular intimal hyperplasia and suggest a novel therapeutic strategy to suppress restenosis by targeting noncanonical MMP-1-PAR1 signaling in vascular SMCs.


Asunto(s)
Estenosis Carotídea/metabolismo , Desdiferenciación Celular , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor PAR-1/metabolismo , Transducción de Señal , Animales , Estenosis Carotídea/genética , Estenosis Carotídea/patología , Estenosis Carotídea/fisiopatología , Estenosis Carotídea/terapia , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Hiperplasia , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/genética , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Miocitos del Músculo Liso/patología , Inhibidores de Proteasas/farmacología , Receptor PAR-1/genética , Túnica Íntima/metabolismo , Túnica Íntima/patología
16.
Blood ; 119(7): 1717-25, 2012 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-22186993

RESUMEN

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.


Asunto(s)
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 Xenoinjerto
17.
Proc Natl Acad Sci U S A ; 108(20): 8491-6, 2011 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-21536878

RESUMEN

Protease-activated receptor-2 (PAR2), a cell surface receptor for trypsin-like proteases, plays a key role in a number of acute and chronic inflammatory diseases of the joints, lungs, brain, gastrointestinal tract, and vascular systems. Despite considerable effort by the pharmaceutical industry, PAR2 has proven recalcitrant to targeting by small molecule inhibitors, which have been unable to effectively prevent the interaction of the protease-generated tethered ligand with the body of the receptor. Here, we report the development of first-in-class cell-penetrating lipopeptide "pepducin" antagonists of PAR2. The design of the third intracellular (i3) loop pepducins were based on a structural model of a PAR2 dimer and by mutating key pharmacophores in the receptor intracellular loops and analogous pepducins. Individual pharmacophores were identified, which controlled constitutive, agonist, and antagonist activities. This approach culminated in the identification of the P2pal-18S pepducin which completely suppressed trypsin and mast cell tryptase signaling through PAR2 in neutrophils and colon cancer cells. The PAR2 pepducin was highly efficacious in blocking PAR2-dependent inflammatory responses in mouse models. These effects were lost in PAR2-deficient and mast-cell-deficient mice, thereby validating the specificity of the pepducin in vivo. These data provide proof of concept that PAR2 pepducin antagonists may afford effective treatments of potentially debilitating inflammatory diseases and serve as a blueprint for developing highly potent and specific i3-loop-based pepducins for other G protein-coupled receptors (GPCRs).


Asunto(s)
Inflamación/tratamiento farmacológico , Lipopéptidos/farmacología , Receptor PAR-2/antagonistas & inhibidores , Animales , Neoplasias del Colon/metabolismo , Inflamación/etiología , Inflamación/prevención & control , Lipopéptidos/síntesis química , Lipopéptidos/uso terapéutico , Mastocitos , Ratones , Neutrófilos , Receptor PAR-2/fisiología , Receptores Acoplados a Proteínas G , Transducción de Señal/efectos de los fármacos , Tripsina/efectos de los fármacos , Triptasas/antagonistas & inhibidores , Triptasas/efectos de los fármacos
18.
J Biol Chem ; 287(16): 12787-96, 2012 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-22374997

RESUMEN

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.


Asunto(s)
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 fármacos
19.
J Biol Chem ; 287(29): 24330-8, 2012 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-22573325

RESUMEN

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.


Asunto(s)
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 Xenoinjerto
20.
Circulation ; 126(1): 83-91, 2012 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-22705889

RESUMEN

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.


Asunto(s)
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/metabolismo
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