Studies on acyl pyrrolidine inhibitors of HCV RNA-dependent RNA polymerase to identify a molecule with replicon antiviral activity

The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta 21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon). (c) 2007 Elsevier Ltd. All rights reserved.

Optimization of novel acyl pyrrolidine inhibitors of hepatitis C virus RNA-dependent RNA polymerase leading to a development candidate

Optimization of a pyrrolidine-based template using structure-based design and physicochemical considerations has provided a development candidate 20b (3082) with submicromolar potency in the HCV replicon and good pharmacokinetic properties.

Identification of GSK625433: A novel clinical candidate for the treatment of hepatitis C.

Hepatitis C is an infection of the liver caused by a pos. single-stranded RNA virus (HCV) which affects 170 million people worldwide. It is responsible for 40-60% of all liver disease and is the major cause of liver transplants in the United States. The HCV NS5B gene encodes the viral RNA-dependent RNA polymerase which is essential for HCV replication. We have previously reported the identification of acylpyrrolidines as potent inhibitors of NS5B; however their activity is attenuated against genotype 1a. The design of improved broader-spectrum compds., capable of effective inhibition of both genotypes 1b and 1a is desirable. An understanding of the binding site and genotype sequence differences was utilized to design compds. with greatly enhanced genotype 1a and 1b potency. Our studies led to the identification of GSK625433, a potent, homochiral inhibitor of these HCV genotypes in both enzyme and sub-genomic replicon cell-based assays. GSK625433 has a good pharmacokinetic profile in pre-clin. animal species, enabling progression to clin. evaluation.

Respiratory viral infection in lower airways of asymptomatic children.

Aim: The aim of this study was to determine if asthmatic children have viruses more commonly detected in lower airways during asymptomatic periods than normal children. Methods: Fifty-five asymptomatic children attending elective surgical procedures (14 with stable asthma, 41 normal controls) underwent non-bronchoscopic bronchoalveolar lavage. Differential cell count and PCR for 13 common viruses were performed. Results: Nineteen (35%) children were positive for at least one virus, with adenovirus being most common. No differences in the proportion of viruses detected were seen between asthmatic and normal ‘control’ children. Viruses other than adenovirus were associated with higher neutrophil counts, suggesting that they caused an inflammatory response in both asthmatics and controls (median BAL neutrophil count, 6.9% for virus detected vs. 1.5% for virus not detected, p = 0.03). Conclusions: Over one-third of asymptomatic children have a detectable virus (most commonly adenovirus) in the lower airway; however, this was not more common in asthmatics. Viruses other than adenovirus were associated with elevated neutrophils suggesting that viral infection can be present during relatively asymptomatic periods in asthmatic children.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wildtype (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs. (C) 2008 Elsevier B.V. All rights reserved.

RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle.

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex

DNA telomeric repeats in mammalian cells are transcribed to guanine-rich RNA sequences, which adopt parallel-stranded G-quadruplexes with a propeller-like fold. The successful crystallization and structure analysis of a bimolecular human telomeric RNA G-quadruplex, folded into the same crystalline environment as an equivalent DNA oligonucleotide sequence, is reported here. The structural basis of the increased stability of RNA telomeric quadruplexes over DNA ones and their preference for parallel topologies is described here. Our findings suggest that the 2'-OH hydroxyl groups in the RNA quadruplex play a significant role in redefining hydration structure in the grooves and the hydrogen bonding networks. The preference for specific nucleotides to populate the C3'-endo sugar pucker domain is accommodated by alterations in the phosphate backbone, which leads to greater stability through enhanced hydrogen bonding networks. Molecular dynamics simulations on the DNA and RNA quadruplexes are consistent with these findings. The computations, based on the native crystal structure, provide an explanation for RNA G-quadruplex ligand binding selectivity for a group of naphthalene diimide ligands as compared to the DNA G-quadruplex.

Selectivity in small molecule binding to human telomeric RNA and DNA quadruplexes

Quadruplex RNAs are less well understood than their DNA counterparts, yet of potentially high biological relevance. The interactions of several quadruplex-binding ligands with telomeric RNA quadruplexes are reported and compared with their binding to the analogous DNA quadruplexes.