RESUMEN
Ribonucleoside phosphonate analogues containing 2'-α-fluoro modifications were synthesized and their potency evaluated against HCV RNA polymerase. The diphosphophosphonate (triphosphate equivalent) adenine and cytidine analogues displayed potent inhibition of the HCV polymerase in the range of 1.9-2.1 µM, but only modest cell-based activity in the HCV replicon. Pro-drugs of the parent nucleoside phosphonates improved the cell-based activity.
Asunto(s)
Antivirales/química , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Flúor/química , Hepacivirus/enzimología , Organofosfonatos/química , Ribonucleósidos/química , Antivirales/síntesis química , Antivirales/farmacología , Línea Celular , ARN Polimerasas Dirigidas por ADN/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the ß-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the ß-hairpin in the thumb subdomain.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Purinas/farmacología , Piridazinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Antivirales/química , Línea Celular , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Mutación , Plásmidos/genética , Purinas/química , Piridazinas/químicaRESUMEN
Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The authors developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5' capped mRNA control. The authors screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1700 initial hits. After secondary counterscreening, the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end, the authors found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, as well we the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.
Asunto(s)
Hepacivirus/genética , Hepacivirus/metabolismo , Ensayos Analíticos de Alto Rendimiento , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína , Animales , Evaluación Preclínica de Medicamentos , Genes Reporteros , Humanos , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Conejos , Reproducibilidad de los Resultados , Proyectos de Investigación , Bibliotecas de Moléculas PequeñasRESUMEN
The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2'-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61-109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC(50)) regardless of genotype or subtype. Importantly, we observed a significant correlation (p<0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.
Asunto(s)
Antivirales/farmacología , Hepacivirus/clasificación , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Línea Celular , Hepatocitos/virología , Humanos , Concentración 50 InhibidoraRESUMEN
BACKGROUND/AIMS: Following lead optimization, a set of substituted imidazopyridines was identified as potent and selective inhibitors of in vitro HCV replication. The particular characteristics of one of the most potent compounds in this series (5-[[3-(4-chlorophenyl)-5-isoxazolyl]methyl]-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine or GS-327073), were studied. METHODS: Antiviral activity of GS-327073 was evaluated in HCV subgenomic replicons (genotypes 1b, 1a and 2a), in the JFH1 (genotype 2a) infectious system and against replicons resistant to various selective HCV inhibitors. Combination studies of GS-327073 with other selective HCV inhibitors were performed. RESULTS: Fifty percent effective concentrations for inhibition of HCV subgenomic 1b replicon replication ranged between 2 and 50 nM and were 100-fold higher for HCV genotype 2a virus. The 50% cytostatic concentrations were > or = 17 microM, thus resulting in selectivity indices of > or = 340. GS-327073 retained wild-type activity against HCV replicons that were resistant to either HCV protease inhibitors or several polymerase inhibitors. GS-327073, when combined with either interferon alpha, ribavirin, a nucleoside polymerase or a protease inhibitor resulted in overall additive antiviral activity. Combinations containing GS-327073 proved highly effective in clearing hepatoma cells from HCV. CONCLUSIONS: GS-327073 is a potent in vitro inhibitor of HCV replication either alone or in combination with other selective HCV inhibitors.
Asunto(s)
Antivirales/farmacología , Hepacivirus/fisiología , Piridinas/farmacología , Replicación Viral/efectos de los fármacos , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Hepacivirus/genética , Humanos , Imidazoles/farmacología , Interferones/farmacología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Inhibidores de Proteasas/farmacología , ARN Viral/metabolismo , Ribavirina/farmacologíaRESUMEN
An efficient and facile synthesis of a large series of diverse 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides (55 examples of both ribo- and 2'-deoxyribonucleosides), aimed at identifying novel homologues of natural nucleosides, was developed. The key transformation involved nucleophilic substitutions of Tol-protected 6-(mesyloxymethyl)purine nucleosides by primary or secondary amines, alcoholates or thiolates. While the 2'-deoxyribonucleosides were inactive, the ribonucleosides exerted considerable cytostatic effects and some anti-HCV activity with low selectivity.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Citostáticos/síntesis química , Citostáticos/farmacología , Hepacivirus/efectos de los fármacos , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Aminación , Animales , Antivirales/química , Línea Celular Tumoral , Citostáticos/química , Humanos , Hidroxilación , Mesilatos/química , Metilación , Ratones , Estructura Molecular , Nucleósidos de Purina/química , Relación Estructura-Actividad , Compuestos de Azufre/síntesis química , Compuestos de Azufre/química , Compuestos de Azufre/farmacologíaRESUMEN
An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic additions of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.
Asunto(s)
Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Alquilación , Aminación , Animales , Antivirales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Humanos , Ratones , Estructura Molecular , Nucleósidos de Purina/química , Relación Estructura-ActividadRESUMEN
A series of purine l-ribonucleosides 2a-2i bearing diverse C-substituents (alkyl, aryl, hetaryl or hydroxymethyl) in the position 6 were prepared by Pd-catalyzed cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-beta-l-ribofuranosyl)purine with the corresponding organometallics followed by deprotection. Unlike their d-ribonucleoside enantiomers that possess strong cytostatic and anti-HCV activity, the l-ribonucleosides were inactive except for 6-benzylpurine nucleoside 2h showing moderate anti-HCV effect in replicon assay. A triphosphate of 2h did not inhibit HCV RNA polymerase.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Ribonucleósidos/síntesis química , Ribonucleósidos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Antivirales/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Células HeLa , Hepacivirus/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nucleósidos de Purina/química , Ribonucleósidos/química , Relación Estructura-ActividadRESUMEN
MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, some of which are known to play important regulatory roles in animals by targeting the messages of protein-coding genes for translational repression. We find that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments diagnostic of miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting productive translation.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Regulación hacia Abajo , Genes Reporteros , Células HeLa , Humanos , Ratones , MicroARNs/química , MicroARNs/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , ARN Mensajero/química , Alineación de Secuencia , Factores de Transcripción/genética , TransfecciónRESUMEN
MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.
Asunto(s)
Regulación de la Expresión Génica/genética , MicroARNs/genética , Interferencia de ARN/fisiología , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Algoritmos , Animales , Artefactos , Biología Computacional/métodos , Evolución Molecular , Marcación de Gen/métodos , Células HeLa , Humanos , Mamíferos , Ratones , Biología Molecular/métodos , Valor Predictivo de las Pruebas , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
The lymphoid-specific proteins RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double-strand breaks at the recombination signal sequences (RSSs). In addition to DNA cleavage, the versatile RAG1/2 complex is capable of catalyzing several other reactions, including hybrid joint formation and the transposition of signal ends into a second DNA. Here we show that the RAG1/2 complex also mediates an unusual strand transfer reaction, inverse transposition, in which non-RSS DNA is cleaved and subsequently transferred to an RSS sequence by direct transesterification. Characterization of the reaction products and requirements suggests that inverse transposition is related to both hybrid joint formation and signal-end transposition. This aberrant activity provides another possible mechanism for some chromosomal translocations present in lymphoid tumors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Reordenamiento Génico/fisiología , Proteínas de Homeodominio/metabolismo , Animales , RatonesRESUMEN
The hepatitis delta virus (HDV) ribozymes are self-cleaving RNA sequences critical to the replication of a small RNA genome. A recently determined crystal structure together with biochemical and biophysical studies provides new insight into the possible catalytic mechanism of these ribozymes. The HDV ribozymes are examples of naturally occurring small ribozymes that catalyze cleavage of the RNA backbone with a rate enhancement of 10(6)- to 10(7)-fold over the uncatalyzed rate. To achieve this level of rate enhancement, the HDV ribozymes have been proposed to employ several catalytic strategies that include the use of metal ions, intrinsic binding energy, and a novel example of general acid-base catalysis with a cytosine side chain acting as a proton donor or acceptor.