RESUMEN
Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Distroglicanos/metabolismo , Laminina/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/metabolismo , Modelos Animales de Enfermedad , Distroglicanos/inmunología , Expresión Génica , Humanos , Inmunohistoquímica , Inyecciones Intramusculares , Laminina/genética , Laminina/inmunología , Ratones , Ratones Noqueados , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Síndrome de Walker-Warburg/tratamiento farmacológico , Síndrome de Walker-Warburg/etiologíaRESUMEN
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 fármacosRESUMEN
Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 µg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Corynebacterium diphtheriae/inmunología , Antitoxina Diftérica/aislamiento & purificación , Antitoxina Diftérica/uso terapéutico , Difteria/prevención & control , Inmunoterapia/métodos , Animales , Línea Celular , Toxina Diftérica/antagonistas & inhibidores , Modelos Animales de Enfermedad , Mapeo Epitopo , Cobayas , Voluntarios Sanos , Humanos , Pruebas de Neutralización , Unión Proteica , Análisis de SupervivenciaRESUMEN
OBJECTIVE: Emerging evidence suggests that protease-activated receptors-1 and -2 (PAR1 and PAR2) can signal together in response to proteases found in the rapidly changing microenvironment of damaged blood vessels. However, it is unknown whether PAR1 and PAR2 promote or mitigate the hyperplastic response to arterial injury. Using cell-penetrating PAR1 pepducins and mice deficient in PAR1 or PAR2, we set out to determine the respective contributions of the receptors to hyperplasia and phenotypic modulation of smooth muscle cells (SMCs) in response to arterial injury. METHODS AND RESULTS: SMCs were strongly activated by PAR1 stimulation, as evidenced by increased mitogenesis, mitochondrial activity, and calcium mobilization. The effects of chronic PAR1 stimulation following vascular injury were studied by performing carotid artery ligations in mice treated with the PAR1 agonist pepducin, P1pal-13. Histological analysis revealed that PAR1 stimulation caused striking hyperplasia, which was ablated in PAR1(-/-) and, surprisingly, PAR2(-/-) mice. P1pal-13 treatment yielded an expression pattern consistent with a dedifferentiated phenotype in carotid artery SMCs. Detection of PAR1-PAR2 complexes provided an explanation for the hyperplastic effects of the PAR1 agonist requiring the presence of both receptors. CONCLUSIONS: We conclude that PAR2 regulates the PAR1 hyperplastic response to arterial injury leading to stenosis.
Asunto(s)
Neointima/patología , Neointima/fisiopatología , Receptor PAR-1/fisiología , Receptor PAR-2/fisiología , Animales , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Proliferación Celular , Femenino , Hiperplasia/patología , Hiperplasia/fisiopatología , Ratones , Ratones Noqueados , Modelos Animales , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Fenotipo , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Receptor PAR-2/deficiencia , Receptor PAR-2/genéticaRESUMEN
Thrombin-dependent platelet activation has been shown to be important in the setting of angioplasty and stenting, which may cause ischemic complications including acute myocardial infarction and death. Inhibitors of the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), are now being evaluated in clinical trials for safety and efficacy in patients with atherothrombotic disease. However, it is unknown whether chronic inhibition of PAR1 in these large patient populations will have beneficial or possibly adverse effects on other biologic processes involved in blood vessel homeostasis and the response to vascular injury. Most recently, PAR1 was found to be cleaved at a distinct site by matrix metalloprotease-1 (MMP-1) to create a longer tethered ligand, which activates a distinct spectrum of G protein pathways in platelets. The differential activation by serine proteases such as thrombin and the metalloprotease MMP-1, places the protease receptor PAR1 at the junction of two major protease classes critically involved in thrombosis, matrix remodeling, and the response to vascular injury.
Asunto(s)
Metaloproteasas/metabolismo , Receptor PAR-1/metabolismo , Serina Proteasas/metabolismo , Transducción de Señal/fisiología , Trombosis/fisiopatología , Lesiones del Sistema Vascular/metabolismo , Animales , Arterias/anatomía & histología , Arterias/metabolismo , Arterias/patología , Activación Enzimática , Humanos , Lesiones del Sistema Vascular/patologíaRESUMEN
Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer.
Asunto(s)
Ascitis/enzimología , Metaloproteasas/antagonistas & inhibidores , Neovascularización Patológica/enzimología , Oligopéptidos/farmacología , Neoplasias Ováricas/patología , Receptor PAR-1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Líquidos Corporales/enzimología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Progresión de la Enfermedad , Docetaxel , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Ratones , Ratones Desnudos , Células 3T3 NIH , Invasividad Neoplásica , Oligopéptidos/administración & dosificación , Oligopéptidos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Peritoneo/enzimología , Peritoneo/patología , Taxoides/administración & dosificación , Taxoides/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The parental conflict, or kinship, theory of genomic imprinting predicts that parent-specific gene expression may evolve in species in which parental investment in developing offspring is unequal. This theory explains many aspects of parent-of-origin transcriptional silencing of embryonic growth regulatory genes in mammals, but it has not been tested in any other live-bearing, placental animals. A major embryonic growth promoting gene with conserved function in all vertebrates is insulin-like growth factor 2 (IGF2). This gene is imprinted in both eutherians and marsupials, as are several genes that modulate IGF2 activity. We have tested for parent-of-origin influences on developmental expression of IGF2 in two poeciliid fish species, Heterandria formosa and Poeciliopsis prolifica, that have evolved placentation independently. We found IGF2 to be expressed bi-allelically throughout embryonic development in both species.