9 research outputs found
A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells
A longstanding belief, generated from the use of transfection reagents to internalize oligonucleotides (ASOs) in cells, is that ASOs exclusively trigger mRNA degradation in the nucleus. In contrast, we have identified a novel cytoplasmic mechanism through which ASOs silence their targets when delivered gymnotically (i.e., in the absence of any transfection reagent). Our data support the existence of a productive, PKC--dependent endocytotic pathway which leads to the interaction of the ASO with the Ago-2 PAZ domain followed by localization of the ASO-Ago2 complex into GW-182 mRNA degradation cytoplasmic bodies (GW-bodies). The degradation products of the targeted mRNA are not generated by Ago-2-directed cleavage and do not seem to exhibit a typical RNase H endonucleolytic cleavage pattern, indicating that other nucleases may be involved. Blocking the evolution of late endosomes into multi-vesicular bodies reduces ASO access to Ago-2 and results in potent reduction of ASO silencing ability. The identification of a cytoplasmic pathway occurring after gymnotic ASO delivery complements the previously known nuclear localization of ASO activity after transfection
Crystallographic Identification of a Noncompetitive Inhibitor Binding Site on the Hepatitis C Virus NS5B RNA Polymerase Enzyme
The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the “thumb” domain, about 30 Å from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization
A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells
A CZTSe solar cell with 8.2% power conversion efficiency fabricated using electrodeposited Cu/Sn/Zn precursor and a three-step selenization process at low Se pressure
A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore
As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity
A global benchmark study using affinity-based biosensors
International audienceTo explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. (C) 2008 Elsevier Inc. All rights reserved