RESUMEN
Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.
Asunto(s)
Clobetasol/farmacología , Miconazol/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Células Madre Pluripotentes/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Cerebelo/patología , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Estratos Germinativos/efectos de los fármacos , Estratos Germinativos/metabolismo , Estratos Germinativos/patología , Humanos , Lisofosfatidilcolinas , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Esclerosis Múltiple/patología , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Fenotipo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores de Glucocorticoides/metabolismo , Regeneración/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.
Asunto(s)
Inhibidores Enzimáticos , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido ras/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/genética , Factores de Intercambio de Guanina Nucleótido ras/metabolismoRESUMEN
All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.
Asunto(s)
Diterpenos/metabolismo , Estimulación Luminosa/efectos adversos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/prevención & control , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de la radiación , Retinaldehído/metabolismo , Animales , Bovinos , Diterpenos/farmacología , Diterpenos/uso terapéutico , Femenino , Masculino , Ratones , Ratones Noqueados , Epitelio Pigmentado de la Retina/efectos de los fármacosRESUMEN
Aspartyl aminopeptidase (DNPEP) has been implicated in the control of angiotensin signaling and endosome trafficking, but its precise biologic roles remain incompletely defined. We performed a high-throughput screen of â¼25,000 small molecules to identify inhibitors of DNPEP for use as tools to study its biologic functions. Twenty-three confirmed hits inhibited DNPEP-catalyzed hydrolysis of angiotensin II with micromolar potency. A counter screen against glutamyl aminopeptidase (ENPEP), an enzyme with substrate specificity similar to that of DNPEP, identified eight DNPEP-selective inhibitors. Structure-activity relationships and modeling studies revealed structural features common to the identified inhibitors, including a metal-chelating group and a charged or polar moiety that could interact with portions of the enzyme active site. The compounds identified in this study should be valuable tools for elucidating DNPEP physiology.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glutamil Aminopeptidasa/antagonistas & inhibidores , Glutamil Aminopeptidasa/metabolismo , Angiotensina II/metabolismo , Animales , Dominio Catalítico/efectos de los fármacos , Humanos , Hidrólisis/efectos de los fármacos , Mamíferos , Células Sf9 , Spodoptera , Relación Estructura-ActividadRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0071093.].
RESUMEN
Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and â¼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.
Asunto(s)
Antineoplásicos/química , Benzamidas/química , Bencenoacetamidas/química , Hidrazinas/química , Indoles/química , Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/química , Sulfonamidas/química , Tetrazoles/química , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Bencenoacetamidas/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrazinas/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Mutación , Invasividad Neoplásica , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Relación Estructura-Actividad , Sulfonamidas/farmacología , Tetrazoles/farmacologíaRESUMEN
PURPOSE: Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS: Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS: Two stabilization assays of P23H mutant opsin were developed and validated, one based on ß-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS: Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.
Asunto(s)
Proteínas Mutantes/genética , Mutación , Retinitis Pigmentosa/genética , Rodopsina/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Immunoblotting , Ratones , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/patologíaRESUMEN
Different diseases of the CNS are associated with blood-brain barrier (BBB) damage and mononuclear cell infiltration. In order to study genes that may play a role in endothelial cell regulation in inflammatory CNS diseases, we performed differential gene expression (DGE) analysis using a mouse brain endothelial cell line. We found that interferon-gamma (IFNgamma)-induced monokine (MIG), a chemokine that plays a role in T lymphocyte and monocyte chemoattraction, is highly expressed in the presence of inflammatory cytokines. We show that MIG, produced by brain endothelial cells in vitro, is biologically active in attractingT lymphocytes and that it is possible to interfere with this mechanism of action using anti-MIG antibodies. We suggest that blocking MIG may be beneficial in CNS inflammation. We detected constitutive expression of the MIG receptor, CXCR3, on the surface of the endothelial cells and therefore hypothesize that it plays a role in maintaining the cytokine gradient at the region of CNS inflammation.
Asunto(s)
Encéfalo/fisiología , Quimiocinas CXC/genética , Endotelio Vascular/fisiología , Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Animales , Encéfalo/citología , Quimiocina CXCL9 , Quimiocinas CXC/fisiología , Quimiotaxis de Leucocito/fisiología , Combinación de Medicamentos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-1/farmacología , Interleucina-2/farmacología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores CXCR3 , Receptores de Quimiocina/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
The ARS Component B gene (EMBL ID: HSARS81S, AC: X99977) encodes a 9 kD non-glycosylated polypeptide (also known as SLURP-1, SwissProt/TrEMBL: P55000), a soluble member of the human Ly6/uPAR superfamily. ARS Component B gene mutations have been implicated in Mal de Meleda. In this study we show by immunohistochemistry that SLURP-1 (secreted Ly-6/uPAR related protein, the protein product of the ARS Component B gene) is localized to human skin, exocervix, gums, stomach and esophagus. In the epidermis, keratinocytes underlying the stratum corneum are highly positive for SLURP1 immunostaining and cultured keratinocytes secrete the expected 9 kD protein. Circulating SLURP1 is detected in human plasma and urine. In the mouse, expression is evident in skin, eye, whole lung, trachea, esophagus and stomach. Human ARS Component B mRNA expression is regulated by retinoic acid, epidermal growth factor and interferon-gamma. The tissue localization and the association with Mal de Meleda suggest that ARS Component B and its protein product SLURP1 are implicated in maintaining the physiological and structural integrity of the keratinocyte layers of the skin.
Asunto(s)
Antígenos Ly/genética , Queratinocitos/metabolismo , Queratodermia Palmoplantar/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Inmunohistoquímica , Queratodermia Palmoplantar/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Estructura Molecular , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Multiple sclerosis, a primary autoimmune disease of the central nervous system has been characterized by the presence of the demyelinating lesions (plaques) in the CNS. To further understand the gene transcription status of the two most common lesions, chronic active and chronic inactive, we have performed a cDNA microarray analysis of these two lesion type. Comparative analysis of differential gene expression of chronic active and inactive lesions have confirmed the existence of a significant difference in the transcriptional profiles of these two lesion types in both marginal and central areas. Different sets of genes were highlighted, including genes of inflammatory characteristics, apoptosis related and stress-induced, indicating their potential role in MS pathogenesis.
Asunto(s)
Perfilación de la Expresión Génica , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Encéfalo/patología , ADN Complementario/genética , Expresión Génica/genética , Silenciador del Gen , Humanos , Persona de Mediana Edad , Esclerosis Múltiple/patología , Reacción en Cadena de la Polimerasa , ARN/genética , Regulación hacia ArribaRESUMEN
Modulation of autotaxin (ATX), the lysophospholipase D enzyme that produces lysophosphatidic acid, with small-molecule inhibitors is a promising strategy for blocking the ATX-lysophosphatidic acid signaling axis. Although discovery campaigns have been successful in identifying ATX inhibitors, many of the reported inhibitors target the catalytic cleft of ATX. A recent study provided evidence for an additional inhibitory surface in the hydrophobic binding pocket of ATX, confirming prior studies that relied on enzyme kinetics and differential inhibition of substrates varying in size. Multiple hits from previous high-throughput screening for ATX inhibitors were obtained with aromatic sulfonamide derivatives interacting with the hydrophobic pocket. Here, we describe the development of a ligand-based strategy and its application in virtual screening, which yielded novel high-potency inhibitors that target the hydrophobic pocket of ATX. Characterization of the structure-activity relationship of these new inhibitors forms the foundation of a new pharmacophore model of the hydrophobic pocket of ATX.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , Sulfonamidas/química , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Relación Estructura-ActividadRESUMEN
High-throughput screening (HTS) of 42 865 compounds was performed to identify compounds that inhibit formation of or kill Staphylococcus epidermidis RP62a biofilms. Three biological processes were assayed, including (1) growth of planktonic/biofilm bacteria, (2) assessment of metabolically active biofilm bacteria using a resazurin assay, and (3) assessment of biofilm biomass by crystal violet staining. After completing the three tiers (primary screening, hit confirmation, and dose-response curves), 352 compounds (representing ~0.8%) were selected as confirmed hit compounds from the HTS assay. The compounds were divided into groups based on their effectiveness on S. epidermidis biofilm properties. The majority of these affected both inhibition and killing of bacterial biofilm cultures. Only 16 of the confirmed hit compounds that have either an AC50 lower than 10 µM and/or Sconst ≥70 from those processed were selected for further study by confocal laser scanning microscopy (CLSM). The CLSM was used to evaluate the confirmed hit compounds on (1) inhibition of biofilm formation and (2) killing of preexisting S. epidermidis biofilms. Taken together, with further testing (e.g., disease-related conditions), such compounds may have applications as broad antimicrobial/antibiofilm use for prophylactic or therapeutic intervention to combat infections in surgical and intensive care clinics and battlefield settings.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana/métodos , Staphylococcus epidermidis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Microscopía Confocal , Bibliotecas de Moléculas Pequeñas , Staphylococcus epidermidis/fisiologíaRESUMEN
Myostatin, a member of the transforming growth factor (TGF)-ß family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-ß family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-ß family.
Asunto(s)
Activinas/antagonistas & inhibidores , Sustancias de Crecimiento/farmacología , Miostatina/antagonistas & inhibidores , Simulación por Computador , Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Bibliotecas de Moléculas PequeñasRESUMEN
Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cumarinas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Experimentales/tratamiento farmacológico , Factores de Crecimiento Nervioso/antagonistas & inhibidores , Tiazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Midkina , Peso Molecular , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Breast and prostate cancers are hormone-sensitive malignancies that afflict millions of women and men. Although prolactin (PRL) is known as a survival factor that supports tumor growth and confers chemoresistance in both cancers, its precise role in these tumors has not been studied extensively. Growth hormone and placental lactogen also bind PRL receptor (PRLR) and mimic some of the actions of PRL. Blockade of the PRLR represents a novel treatment for patients with advanced breast or prostate cancer with limited therapeutic options. This review discusses different approaches for generating PRLR antagonists. Emphasis is placed on technological advances which enable high-throughput screening for small molecule inhibitors of PRLR signaling that could serve as oral medications.
Asunto(s)
Neoplasias de la Mama/terapia , Carcinoma/terapia , Terapia Molecular Dirigida/métodos , Neoplasias de la Próstata/terapia , Receptores de Prolactina/antagonistas & inhibidores , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Ensayos Analíticos de Alto Rendimiento/métodos , Antagonistas de Hormonas/aislamiento & purificación , Antagonistas de Hormonas/uso terapéutico , Humanos , Masculino , Modelos Biológicos , Modelos Moleculares , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores de Prolactina/química , Receptores de Prolactina/metabolismo , Receptores de Prolactina/fisiología , Terapias en Investigación/métodosRESUMEN
PURPOSE: The aim of this study is to test the predictive power of in vivo multiorgan RNA expression profiling in identifying the biologic activity of molecules. METHODS: Animals were treated with compound A or B. At the end of the treatment period, in vivo multiorgan microarray-based gene expression data were collected. Investigators masked to the identity of the compounds analyzed the transcriptome signatures to define the molecular pathways affected by treatment and to hypothesize the biologic activity and potential therapeutic indications of the blinded compounds. RESULTS: For compound A, G-protein-coupled receptors and factors associated with cell growth were affected-growth hormone/insulin-like growth factor-1, glucagon/insulin axes, and general somatomedin-like activity. Deblinding showed the compound to be a somatostatin analog, SOM230, confirming the accuracy of the predicted biologic activity. For compound B, components of the inflammatory cascade potentially mediated by lipopolysaccharide, tumor necrosis factor, or proinflammatory cytokines were affected. The gene expression signatures were most consistent with an interleukin-6 family activity. Deblinding revealed that compound B was leukemia inhibitory factor. CONCLUSIONS: VeloceGenomics is a strategy of coupling in vivo compound testing with genomic technologies. The process enables prediction of the mechanism of action and, coupled with other relevant data, prediction of the suitability of compounds for advancement in the drug development process.
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Diseño de Fármacos , Farmacogenética/métodos , Proteínas/fisiología , Animales , Femenino , Expresión Génica/efectos de los fármacos , Interleucina-6/farmacología , Factor Inhibidor de Leucemia , Macaca fascicularis , Masculino , Valor Predictivo de las Pruebas , Análisis por Matrices de Proteínas , Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Somatostatina/análogos & derivados , Somatostatina/farmacología , Relación Estructura-ActividadRESUMEN
cDNA microarray analysis of the regions of pathologically proven different activity of multiple sclerosis lesions was performed. Major differences in gene expression (DGE) occurred between the lesion margin and lesion centre in active lesions studied (57 and 69 genes differentially expressed, respectively), whereas the margins and centres of silent lesions showed markedly reduced heterogeneity (only 11 and two genes differentially expressed, respectively). To compare differences between chronic active and silent lesions, we performed DGE comparison of the pooled data from both types of lesions. The major DGE occurred at the lesion margin, 156 (26; 5%), the greater number representing upregulated genes at the margin of active lesions (15%). Fourteen genes were found to be significantly upregulated in marginal versus central zones in active lesions examined. These genes comprised predominantly inflammation/immune-related factors. We also performed DGE analysis of pooled genes upregulated at the margin of active lesions and found that among the 50 genes showing differences, nine out of 14 were identified in the previous analysis of overlapping differentially expressed genes. Thus this microarray analysis has identified a novel set of genes associated with lesion activity in multiple sclerosis, many of them not previously linked with the disease.
Asunto(s)
Encéfalo/metabolismo , ADN Complementario/análisis , Perfilación de la Expresión Génica , Esclerosis Múltiple/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas de Ciclo Celular/genética , Enfermedad Crónica , Citocinas/genética , Regulación de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , Esclerosis Múltiple/inmunología , Receptores de Superficie Celular/genética , Sistemas de Mensajero Secundario/genética , Factores de Transcripción/genéticaRESUMEN
We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.