11 research outputs found
Fall Risk Factors for Community-Dwelling Older Adults: An Evidence-Based Practice Project
What are the descriptive or predictive characteristics of community-dwelling older adults who are most at risk for falls
The HCV Non-Nucleoside Inhibitor Tegobuvir Utilizes a Novel Mechanism of Action to Inhibit NS5B Polymerase Function
<div><p>Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays <em>in vitro</em>, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.</p> </div
Risk of radiation-associated intracranial malignancy after stereotactic radiosurgery: a retrospective, multicentre, cohort study
Western analysis of NS5B treated with biotinylated TGV analog shows presence of compound in faster migrating component of double band.
<p>A and B Top) Anti-NS5B Western blot of lysates from DMSO-, TGV-, and biotin compound-treated A) Δ21 NS5B overexpressing cells and B) 1b (Con-1) replicon cells. A and B Middle) Fluorochrome-linked streptavidin detection of biotinylated compound 3. A and B Bottom) Merge of NS5B and biotin detection reveals signal co-localization of faster migrating NS5B species and compound.</p
Overexpression of CYP1A1 in SL3 (HeLa GT 1b) replicon cells restores sensitivity to TGV.
<p>A) qPCR analysis of CYP1A1, 1A2, and 3A4 mRNA levels in 1b Rluc replicon, control SL3 HeLa 1b replicon cells, and SL3 HeLa 1b replicon CYP1A1-overexpressing cell lines. B) TGV, HCV-796, and 2′CMeA EC<sub>50</sub>s in SL3 control and CYP overexpressing cell lines.</p
Reduction of GSH by BSO alters TGV potency and NS5B double band formation.
<p>A) EC<sub>50</sub>s of TGV and VX-222 in 1b replicon cells co-treated with BSO. Replicon cells were pretreated for 24 hours with 5 mM BSO to reduce GSH prior to compound addition. B) NS5B Western blot of lysates from full-length NS5B overexpressing Lunet cells treated varying concentrations of BSO added together with 10 µM TGV.</p
NS5B double band formation in TGV-treated NS5B overexpressing cells.
<p>A) NS5B Western blot of lysates collected from TGV-treated Lunet cells expressing full-length NS5B via a BacMam system. Overexpressing cells were treated for 16 hours with various concentrations of TGV and compared to lysates from replicon cells treated for 24 hours with 50 nM TGV or DMSO as a control. B) Western analysis of TGV-treated cells overexpressing a 21 amino acid C-terminally truncated NS5B (Δ21). C) Immunoprecipitation and Western blots of overexpressed Δ21 NS5B encoding an N- or C-terminal His tag. NS5B Western blotting of anti-His immunoprecipitated material and His Western blotting of anti-NS5B immunoprecipitated material are shown.</p
TGV treatment leads to formation of an NS5B double band detectable by Western analysis.
<p>A) NS5B, NS5A, and NS3 Western blots of lysates from 1b (Con-1) replicon cells treated with 50 nM TGV for various periods of time. B) NS5B Western blot of lysates from 1b replicon cells treated for 24 hours with known HCV inhibitors at 50× EC<sub>50</sub> concentrations. C) NS5B Westerns of cell lysates collected at various times from Huh-luc 1a replicon cells treated with 50 nM or 500 nM TGV and lysates from HeLa 1b replicon cells treated with 10 µM TGV. D) TGV EC<sub>50</sub> values in Huh-luc 1a, 1b, and HeLa 1b (clone SL3) replicon cell lines. E) Western blot of lysates from Lunet cells transiently transfected with wild-type or NS5B Y448H mutant 1b Pi-Rluc replicons and treated with varying concentrations of TGV for 24 hours.</p