Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bortezomib/uso terapéutico , Dexametasona/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Oligorribonucleótidos/antagonistas & inhibidores , Oligorribonucleótidos/uso terapéutico , Anciano , Quimiocina CXCL12 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
As an obligate step for picornaviruses to replicate their genome, the small viral peptide VPg must first be specifically conjugated with uridine nucleotides at a conserved tyrosine hydroxyl group. The resulting VPg-pUpU serves as the primer for genome replication. The uridylylation reaction requires the coordinated activity of many components, including the viral polymerase, a conserved internal RNA stem loop structure, and additional viral proteins. Formation of this complex and the resulting conjugation reaction catalyzed by the polymerase, offers a number of biochemical targets for inhibition of an essential process in the viral life cycle. Therefore, an assay recapitulating uridylylation would provide multiple opportunities for discovering potential antiviral agents. Our goal was to identify inhibitors of human rhinovirus (HRV) VPg uridylylation, which might ultimately be useful to reduce or prevent HRV-induced lower airway immunologic inflammatory responses, a major cause of asthma and chronic obstructive pulmonary disease exacerbations. We have reconstituted the complex uridylylation reaction in an AlphaScreen suitable for high-throughput screening, in which a rabbit polyclonal antiserum specific for uridylylated VPg serves as a key reagent. Assay results were validated by quantitative mass spectrometric detection of uridylylation.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Oligorribonucleótidos/genética , Uridina/genética , Genoma Viral , Humanos , Oligorribonucleótidos/antagonistas & inhibidores , ARN Viral/genética , Tirosina/genética , Uridina/antagonistas & inhibidores , Replicación ViralRESUMEN
Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5'â3' and 3'â5' processes, but the relative contributions are not known. The 3' end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3' UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of â¼40 min with the uncleavable cap compared to â¼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5'â3' decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5' decay intermediates were oligouridylated. Preventing oligouridylation by 3'-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3'â5' degradation machinery stalls during initial degradation, whereupon reuridylation occurs.
Asunto(s)
Oligorribonucleótidos/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Nucleótidos de Uracilo/metabolismo , Regiones no Traducidas 3'/fisiología , Replicación del ADN/efectos de los fármacos , Desoxiadenosinas/farmacología , Silenciador del Gen , Células HeLa , Histonas/metabolismo , Humanos , Hidroxiurea/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oligorribonucleótidos/antagonistas & inhibidores , Polinucleotido Adenililtransferasa , Biosíntesis de Proteínas , Estabilidad del ARN/genética , ARN Mensajero/química , Transducción Genética , Nucleótidos de Uracilo/antagonistas & inhibidores , Factores de Escisión y Poliadenilación de ARNmRESUMEN
The 2-5A system (2-5OAS/RNaseL) is composed of the 2',5'oligoadenylate synthetase 1 (2-5OAS1) and 2-5A-dependent RNase (RNaseL), enzymes that play a key role in antiviral defence mechanisms. Activation of the 2-5A system by double stranded RNA (dsRNA) induces degradation of ribosomal RNAs and apoptosis in mammalian cells. To obtain further information into the molecular mechanisms by which RNaseL induces apoptosis, we expressed human RNaseL and 2-5OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis. In this expression virus-cell system the activation of RNaseL, as index of rRNA degradation, is an upstream event of apoptosis induction. RNaseL induces apoptosis in a caspase-dependent manner (caspases 8, 9 and 2). At the beginning of apoptosis RNaseL and 2-5OAS are localized in the mitochondria and cytosol fractions, while at the onset of apoptosis both enzymes are largely in mitochondria. The 2-5A system induces the release of Cytochrome c from mitochondria to cytosol in a caspase dependent manner. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (delta psi m), as well as the generation of reactive oxygen species (ROS). Moreover, the activation of RNaseL induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of delta psi m induced by the activation of RNaseL. These findings provide new insights into the molecular mechanisms of apoptosis induction by the 2-5A system, demonstrating the importance of mitochondria in 2-5OAS/RNaseL-induced apoptosis.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Apoptosis/fisiología , Mitocondrias/fisiología , Oligorribonucleótidos/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Nucleótidos de Adenina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Citocromos c/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Riñón/citología , Potenciales de la Membrana/fisiología , Mitocondrias/patología , Modelos Biológicos , Oligorribonucleótidos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , ARN Viral/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Virus Vaccinia/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoAsunto(s)
Anticoagulantes/antagonistas & inhibidores , Anticoagulantes/farmacología , Antídotos/farmacología , Diseño de Fármacos , Factor IXa/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , ARN/farmacología , Anticoagulantes/metabolismo , Antídotos/metabolismo , Contraindicaciones , Factor IXa/metabolismo , Factor VIIIa/metabolismo , Factor X/metabolismo , Heparina/farmacología , Antagonistas de Heparina/farmacología , Humanos , Oligorribonucleótidos/antagonistas & inhibidores , Oligorribonucleótidos/metabolismo , ARN/antagonistas & inhibidores , ARN/metabolismo , Especificidad por Sustrato , Trombina/metabolismo , Trombocitopenia/inducido químicamenteRESUMEN
Many therapeutic agents are associated with adverse effects in patients. Anticoagulants can engender acute complications such as significant bleeding that increases patient morbidity and mortality. Antidote control provides the safest means to regulate drug action. For this reason, despite its known limitations and toxicities, heparin use remains high because it is the only anticoagulant that can be controlled by an antidote, the polypeptide protamine. To date, no generalizable strategy for developing drug-antidote pairs has been described. We investigated whether drug-antidote pairs could be rationally designed by taking advantage of properties inherent to nucleic acids to make antidote-controlled anticoagulant agents. Here we show that protein-binding oligonucleotides (aptamers) against coagulation factor IXa are potent anticoagulants. We also show that oligonucleotides complementary to these aptamers can act as antidotes capable of efficiently reversing the activity of these new anticoagulants in plasma from healthy volunteers and from patients who cannot tolerate heparin. This generalizable strategy for rationally designing a drug-antidote pair thus opens up the way for developing safer regulatable therapeutics.
Asunto(s)
Anticoagulantes/antagonistas & inhibidores , Anticoagulantes/farmacología , Antídotos/farmacología , Diseño de Fármacos , Factor IXa/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , ARN/farmacología , Anticoagulantes/química , Anticoagulantes/metabolismo , Antídotos/química , Antídotos/metabolismo , Secuencia de Bases , Unión Competitiva/efectos de los fármacos , Contraindicaciones , Factor IXa/metabolismo , Factor VIIIa/metabolismo , Factor X/metabolismo , Heparina/farmacología , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/antagonistas & inhibidores , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , ARN/antagonistas & inhibidores , ARN/genética , ARN/metabolismo , Trombocitopenia/inducido químicamenteRESUMEN
Respiratory syncytial virus (RSV) continues as an emerging infectious disease not only among infants and children, but also for the immune-suppressed, hospitalized and the elderly. To date, ribavirin (Virazole) remains the only therapeutic agent approved for the treatment of RSV. The prophylactic administration of palivizumab is problematic and costly. The quest for an efficacious RSV antiviral has produced a greater understanding of the viral fusion process, a new hypothesis for the mechanism of action of ribavirin, and a promising antisense strategy combining the 2'-5' oligoadenylate antisense (2-5A-antisense) approach and RSV genomics.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/antagonistas & inhibidores , Nucleótidos de Adenina/metabolismo , Adulto , Anciano , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antivirales/farmacología , Niño , Preescolar , Diseño de Fármacos , Endorribonucleasas/metabolismo , Humanos , Lactante , Interferones/fisiología , Fusión de Membrana/efectos de los fármacos , Ratones , Persona de Mediana Edad , Estructura Molecular , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Oligorribonucleótidos/antagonistas & inhibidores , Oligorribonucleótidos/metabolismo , Palivizumab , ARN Viral/antagonistas & inhibidores , Infecciones por Virus Sincitial Respiratorio/terapia , Ribavirina/análogos & derivados , Ribavirina/química , Ribavirina/farmacología , Ribavirina/uso terapéutico , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/químicaRESUMEN
The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Nucleótidos de Adenina/antagonistas & inhibidores , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , VIH-1/fisiología , Oligorribonucleótidos/antagonistas & inhibidores , Línea Celular , Regulación hacia Abajo , Endorribonucleasas/metabolismo , Humanos , Transfección , Replicación ViralRESUMEN
In vitro studies have shown that the translational inhibitory activity of 2-5 A can be blocked by the oligoribonucleotide 2',5'-(pA)3. We have examined the effect of simultaneous introduction of inhibitor and antagonist into intact mouse cells using calcium phosphate coprecipitation. Upon introduction of 10(-4) M 2',5'-(pA)3 and 10(-6) M 2-5 A, inhibition of protein synthesis was prevented. Efficiency of calcium phosphate precipitation of 2-5 A in the presence or absence of 2',5'-(pA)3 was comparable. Introduction of 2',5'-(pA)3 analogs showed that nucleotides which do not bind well to the 2-5 A dependent endonuclease do not prevent 2-5 A inhibitory activity. Thus, 2',5'-(pA)3 functions as an antagonist of 2-5 A in vivo.
Asunto(s)
Nucleótidos de Adenina/antagonistas & inhibidores , Nucleótidos de Adenina/farmacología , Oligorribonucleótidos/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , Biosíntesis de Proteínas , Animales , Células L/metabolismo , RatonesRESUMEN
Chemically synthesised CH3Sp(A2'p)2A2'pp3'OCH3 has been used to assess the importance of the ppp(A2'p)nA (n greater than or equal to 2: 2-5A) system in the antiviral action of interferon against encephalomyocarditis virus (EMC). It inhibits activation of the 2-5A-dependent RNase by 2-5A in intact mouse L929 cells and cell-free systems. In interferon-treated, EMC-infected L929 cells it inhibits 2-5A-mediated rRNA cleavage and partially restores EMC RNA synthesis and virus yield. Activation of the 2-5A-dependent RNase must, therefore, play some part in interferon action against the growth of EMC virus in such cells.