RESUMEN
Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pKa. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.
Asunto(s)
Amidas/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Bazo/enzimología , Amidas/síntesis química , Amidas/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Pruebas de Mutagenicidad , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Bazo/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Pigment epithelium-derived factor (PEDF) can delay and prevent the death of photoreceptors in vivo. We investigated the survival activity of PEDF on cone photoreceptor-derived 661W cells in culture, the presence of PEDF receptor (PEDF-R) in these cells and the activation of prosurvival Akt. Cell death was induced by light exposure in the presence of 9-cis retinal. Cell viability assays showed that PEDF increased the number of 661W cells exposed to these conditions. Western blots showed that PEDF-treated 661W cells had a higher ratio of phosphorylated Akt to total Akt than untreated cells. The PEDF receptor PEDF-R was immunodetected in the plasma membrane fractions of 661W cells. The results demonstrated that PEDF can protect 661W cells against light-induced cell death and suggest that the binding of PEDF to cell surface PEDF-R triggers a prosurvival signaling pathway.
Asunto(s)
Proteínas del Ojo/metabolismo , Luz/efectos adversos , Factores de Crecimiento Nervioso/metabolismo , Fármacos Neuroprotectores/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Fotorreceptoras Retinianas Conos/fisiología , Serpinas/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Muerte Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Proteínas del Ojo/farmacología , Ratones , Factores de Crecimiento Nervioso/farmacología , Fármacos Neuroprotectores/farmacología , Fosforilación/fisiología , Receptores de Neuropéptido/metabolismo , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Serpinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.
Asunto(s)
ADN Mitocondrial/genética , Células Epiteliales/metabolismo , Expresión Génica , Células Híbridas/metabolismo , Degeneración Macular/genética , Mitocondrias/genética , Transducción de Señal/genética , Adenosina Trifosfato/biosíntesis , Células Cultivadas , Complemento C3/genética , Complemento C3/metabolismo , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , ADN Mitocondrial/metabolismo , Células Epiteliales/citología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Haplotipos , Humanos , Células Híbridas/patología , Ácido Láctico/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Mitocondrias/metabolismo , Modelos Biológicos , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Intramolecular inverse electron demand cycloadditions of isatin-derived 1,2,4-triazines with acetylenic dienophiles tethered by amidations or transesterifications proceed in excellent yields to produce lactam- or lactone-fused α-carbolines. Beginning with various isatins and alkynyl dienophiles, a pilot-scale library of eighty-eight α-carbolines was prepared by using this robust methodology for biological evaluation.
RESUMEN
The chemistry of 1,2,3,4-tetrahydro-1,5-naphthyridines and 2,3,4,5-tetrahydro-1H-pyrido[3,2-b]azepines has been explored with the goal of discovering reactions at N1 suitable for library development. Epoxide openings, palladium-catalyzed N-arylations, DEPBT-promoted acylations, and urea formation through the reaction with isocyanates were all successful. The epoxide opening chemistry using homochiral epichlorohydrin, with epoxide reclosure and a second nucleophilic opening led to the preparation of a small 24-membered library.
RESUMEN
A primary limitation in the development and use of screens to identify factors that regulate mammalian pre-mRNA splicing has been the development of sensitive reporter assays. Alternative splicing typically involves relatively small (< 10-fold) changes in isoform ratios. Therefore, reporter constructs designed to allow direct analysis of isoform expression historically have at most a 10-fold window of discrimination between a positive signal and background. Here we describe the design and application of a reporter cell line that makes use of the phenomenon of transcriptional synergy to amplify the detection of changes in splicing, such that a three- to five-fold change in splicing pattern is observed as a 30- to 50-fold change in GFP expression. Using this cell line we have identified two small molecules, from a library of approximately 300 synthetic compounds, that can induce partial repression of a variable exon from the CD45 gene. We propose that the concept of transcription-based amplification of signal will allow the development of true high-throughput screening approaches to identify effectors of mammalian alternative splicing.
Asunto(s)
Empalme Alternativo , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Empalme del ARN , Transcripción Genética , Línea Celular , Exones , Biblioteca de Genes , Antígenos Comunes de Leucocito/efectos de los fármacos , Antígenos Comunes de Leucocito/genética , Proteínas Luminiscentes/efectos de los fármacos , Proteínas Luminiscentes/genética , Activación de Linfocitos/efectos de los fármacos , Precursores del ARN/metabolismo , Sensibilidad y Especificidad , Transducción de Señal , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
The intramolecular inverse-electron-demand Diels-Alder reaction between imidazoles and 1,2,4-triazines linked by a trimethylene tether from the imidazole N1 position to the triazine C3 proceed in excellent yields to produce 1,2,3,4-tetrahydro-1,5-naphthyridines. The reaction proceeds by a cycloaddition with subsequent loss of nitrogen, followed by a presumed stepwise loss of a nitrile. The analogous intramolecular cycloadditions employing a tetramethylene tether also proceeded to give 2,3,4,5-tetrahydro-1H-pyrido[3,2-b]azepines in acceptable yields. The reaction to produce the tetrahydro-1,5-naphthyridines can also be promoted with microwave irradiation.
Asunto(s)
Electrones , Compuestos Heterocíclicos/síntesis química , Imidazoles/síntesis química , Naftiridinas/síntesis química , Triazinas/química , Ciclización , Imidazoles/química , Estructura MolecularRESUMEN
The study discussed in this article addressed the relationship of social support to the maintenance of long-term safer sex practices of 360 HIV-positive adults recruited from outpatient medical facilities. Medical professionals, friends, and siblings were reported the most frequent sources for assistance, whereas regular sexual partners, medical professionals, and community organizations were rated as the most helpful. Differences in social support use across ethnic, gender, and sexual orientation groups were observed. Those engaging in safer sex practices perceived the support they received as more helpful. These findings emphasize the link between positive support networks and avoidance of high-risk sexual behavior in HIV-positive individuals. Implications for the delivery of culturally appropriate, gender-specific, and community-based interventions are discussed.
Asunto(s)
Infecciones por VIH/psicología , Sexo Seguro/psicología , Sexualidad/psicología , Apoyo Social , Adulto , Análisis de Varianza , Femenino , Infecciones por VIH/etnología , Infecciones por VIH/transmisión , Conductas Relacionadas con la Salud/etnología , Encuestas Epidemiológicas , Humanos , Masculino , Persona de Mediana Edad , Nevada , Religión y Psicología , Sexo Seguro/etnología , Factores Sexuales , Sexualidad/clasificación , Sexualidad/etnologíaRESUMEN
Access to services and their relationship to the maintenance of long-term safer sex practices are addressed in this study of 360 HIV+ adults recruited from outpatient medical facilities. Protease inhibitors, antiviral therapies, and entitlements were reported as the most needed services, while entitlements and money to pay for housing were reported as the largest unmet needs. Differences across ethnic and gender groups were observed. One-third of all respondents reported at least one occasion of unprotected anal or vaginal intercourse in the previous six months. The practice of unsafe sex was found to be significantly related to both the number of needed services and the number of unmet needs, even after controlling for demographic variables. In addition, a higher proportion of those who engaged in unsafe sex reported a higher need for psychological counseling and social support. These findings underscore the important linkage between access to services with avoidance of high-risk sexual behavior in HIV+ persons. Implications for the delivery of culturally appropriate, gender-specific and community-based interventions are discussed.