RESUMEN
The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.
Asunto(s)
Benzopiranos/síntesis química , Quinolinas/síntesis química , Receptores de Glucocorticoides/efectos de los fármacos , Animales , Benzopiranos/química , Benzopiranos/farmacología , Unión Competitiva , Carragenina , Línea Celular , Chlorocebus aethiops , Depresión Química , Selectina E/genética , Selectina E/metabolismo , Edema/inducido químicamente , Edema/patología , Eosinófilos/patología , Genes Reporteros , Humanos , Insectos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Neumonía/patología , Quinolinas/química , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/genética , Elementos de Respuesta , Relación Estructura-Actividad , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
High-throughput screening for the induction of a luciferase reporter gene in a thrombopoietin (TPO)-responsive cell line resulted in the identification of 4-diazo-3-hydroxy-1-naphthalenesulfonic acids as TPO mimics. Modification of the core structure and adjustment of unwanted functionality resulted in the development of (5-oxo-1,5-dihydropyrazol-4-ylidene)hydrazines which exhibited efficacies equivalent to those of TPO in several cell-based assays designed to measure thrombopoietic activity. Furthermore, these compounds elicited biochemical responses in TPO-receptor-expressing cells similar to those in TPO itself, including kinase activation and protein phosphorylation. Potencies for the best compounds were high for such low molecular weight compounds (MW < 500) with EC(50) values in the region of 1-20 nM.
Asunto(s)
Compuestos Azo/síntesis química , Hidrazinas/síntesis química , Megacariocitos/efectos de los fármacos , Naftalenosulfonatos/síntesis química , Proteínas de Neoplasias , Pirazoles/síntesis química , Receptores de Citocinas , Trombopoyetina/química , Animales , Compuestos Azo/química , Compuestos Azo/farmacología , División Celular , Línea Celular , Evaluación Preclínica de Medicamentos , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Genes Reporteros , Hidrazinas/química , Hidrazinas/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Imitación Molecular , Peso Molecular , Naftalenosulfonatos/química , Naftalenosulfonatos/farmacología , Fosforilación , Fosfotransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Receptores de Trombopoyetina , Relación Estructura-Actividad , Trombopoyetina/metabolismoRESUMEN
A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).
Asunto(s)
Antiinflamatorios/síntesis química , Benzopiranos/síntesis química , Prednisolona/farmacología , Quinolinas/síntesis química , Receptores de Glucocorticoides/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Benzopiranos/química , Benzopiranos/metabolismo , Benzopiranos/farmacología , Línea Celular , Selectina E/genética , Selectina E/metabolismo , Genes Reporteros , Humanos , Ligandos , Luciferasas/genética , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacología , Ratas , Estereoisomerismo , Activación Transcripcional , TransfecciónRESUMEN
Drug discovery using intracellular receptors (IRs) as targets presents its own set of unique complications and advantages. The natural ligands for these receptors are, in many cases, already used as drugs. To effectively exploit these targets, newer molecules must have either increased receptor selectivity or increased tissue or gene selectivity to reduce side effects. The search for these molecules will yield new therapeutics as well as new insights into the mechanism of action of these receptors and their ligands.
Asunto(s)
Diseño de Fármacos , Ligandos , Receptores de Superficie Celular/metabolismo , Animales , Células Cultivadas , Humanos , Modelos MolecularesRESUMEN
Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.
Asunto(s)
Replicación del ADN/genética , Herpesvirus Humano 4/genética , Plásmidos/genética , Replicón/genética , Bromodesoxiuridina/metabolismo , Línea Celular , Metilación de ADN , Enzimas de Restricción del ADN/metabolismo , Proteínas de Unión al ADN/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Humanos , Origen de Réplica/genética , Transcripción Genética/genética , Transfección/genética , Proteínas Virales/genéticaRESUMEN
To study the mechanism of basal transcription by RNA polymerase II, a cDNA encoding the Drosophila homologue of the small subunit of TFIIF (also referred to as TFIIF30, RAP30, factor 5b, and gamma) was isolated. The Drosophila TFIIF30 gene is located at region 86C on the right arm of the third chromosome. The protein encoded by the cDNA, termed dTFIIF30, was synthesized in Escherichia coli and purified to greater than 95% homogeneity. In reconstituted transcription reactions with purified basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, designated dF30(119-276), which contains the carboxyl-terminal 158 amino acid residues of dTFIIF30, was found to possess approximately 50% of the transcriptional activity as full-length dTFIIF30. The interaction of dTFIIF30 with the large subunit of TFIIF (also referred to as TFIIF74, RAP74, factor 5a, and beta) was investigated by glycerol gradient sedimentation analyses. In these experiments, dTFIIF30, but not dF30(119-276), assembled into a stable heteromeric complex with TFIIF74. These results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxylterminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.
Asunto(s)
Drosophila melanogaster/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Drosophila melanogaster/genética , Escherichia coli , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , XenopusRESUMEN
Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Genes de Insecto , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Polimerasa II/metabolismo , TATA Box , Proteína de Unión a TATA-Box , Transcripción GenéticaRESUMEN
In eukaryotes, initiation of mRNA synthesis is a multistep process that is carried out by RNA polymerase II and auxiliary factors that are commonly referred to as basal or general factors. In this study accurate initiation of transcription was reconstituted with purified, Escherichia coli-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP30), along with purified, native RNA polymerase II from Drosophila embryos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both subunits of TFIIE and the 74-kD subunit of TFIIF increased the efficiency of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombinant TBP resulted in a strong dependence on TFIIE. By gel mobility-shift analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble into DB and DBPolF30 complexes with transcriptionally competent (wild type or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcription subsequent to assembly of the DBPolF30 complex, which is a minitranscription complex that represents the central core of the RNA polymerase II transcriptional machinery.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Drosophila/enzimología , Drosophila/genética , Escherichia coli/enzimología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , TATA Box , Proteína de Unión a TATA-Box , Moldes Genéticos , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción TFIIB , Factor de Transcripción TFIID , Factores de Transcripción/genéticaRESUMEN
We describe the preparation and biochemical properties of a soluble nuclear fraction derived from Drosophila embryos. This extract, which can be easily prepared in 2.5 hr, is capable of accurate and efficient RNA polymerase II transcription of a variety of diverse genes from both Drosophila and mammals. With the relatively strong promoter of the Drosophila Krüppel gene, it is possible to achieve 20% template usage in a single round of transcription, which is considerably higher than the template usage of approximately 3% seen with standard nuclear extracts. Further, although U small nuclear RNA genes are refractory to transcription with HeLa transcription extracts, the soluble nuclear fraction transcribes a U1 small nuclear RNA gene from Drosophila. Moreover, transcriptional activation by sequence-specific activators can be attained in vitro with the soluble nuclear fraction. The overall transcriptional efficiency appears limited to 0.45 transcript per template of DNA per 30 min, but the mechanism of limitation is not known. The soluble nuclear fraction, which was developed to recreate the environment within the nucleus, should be useful when high efficiencies of RNA polymerase II transcription are desired.
Asunto(s)
Núcleo Celular/metabolismo , Drosophila/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila/embriología , Embrión no Mamífero/metabolismo , Genes , Cinética , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genéticaRESUMEN
We have subdivided the components of the basic RNA polymerase II machinery from Drosophila embryos into three fractions and RNA polymerase II. The RNA polymerase II was 90% homogeneous and possessed the IIa form of the largest subunit. By substitution of these factors with their mammalian homologues in reconstituted transcription reactions, we have determined that the three Drosophila fractions contain transcription factor (TF)IIB, TFIIE/F, and TFIID. In addition, the fraction with TFIID contains another essential activity, which we have tentatively designated as TFIIZ. There was no apparent requirement for TFIIA. The reconstituted transcription factors accurately transcribe both Drosophila and mammalian genes. This fractionated system should serve as a source of general transcription factors for the study of RNA polymerase II transcription in Drosophila as well as other eukaryotes.
Asunto(s)
Drosophila/enzimología , ARN Polimerasa II/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , Animales , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Drosophila/embriología , Drosophila/genética , Embrión no Mamífero/enzimología , Sustancias Macromoleculares , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Human manganese poisoning or manganism results in damage to the substantia nigra of the brain stem, a drop in the level of the inhibitory neurotransmitter dopamine, and symptoms resembling those of Parkinson's disease. Manganic (Mn3+) manganese ions were shown to be readily produced by O-2 in vitro and spontaneously under conditions obtainable in the human brain. Mn3+ as its pyrophosphate complex was shown to rapidly and efficiently carry out four-electron oxidations of dopamine, its precursor dopa (3,4-dihydroxyphenylalanine), and its biosynthetic products epinephrine and norepinephrine. Mn3+-pyrophosphate was shown to specifically attack dihydroxybenzene derivatives, but only those with adjacent hydroxyl groups. Further, the addition of Mn2+-pyrophosphate to a system containing a flux of O2- and dopamine greatly accelerated the oxidation of dopamine. The oxidation of dopamine by Mn3+ neither produced nor required O2, and Mn3+ was far more efficient than Mn2+, Mn4+ (MnO2), O2-, or H2O2 in oxidizing the catecholamines. A higher oxidation state, Mn(OH)3, formed spontaneously in an aqueous Mn(OH)2 precipitate and slowly darkened, presumably being oxidized to MnO2. Like reagent MnO2, it weakly catalyzed dopamine oxidation. However, both MnO2 preparations showed dramatically increased abilities to oxidize dopamine in the presence of pyrophosphate due to enhancement of the spontaneous formation of the Mn3+ complex. These results strongly suggest that the pathology of manganese neurotoxicity is dependent on the ease with which simple Mn3+ complexes are formed under physiological conditions and the efficiency with which they destroy catecholamines.