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NS5B
About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.
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TL;DR: It is demonstrated 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.
Abstract: 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 in vitro, 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.
43 citations
TL;DR: NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle.
Abstract: We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2',4'-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 μM). 2',4'-diF-rU/C represents rare examples of "locked" ribonucleoside mimics that lack a bicyclic ring structure.
43 citations
TL;DR: The cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death and certain HCV genomic structures may determine the viral replication capacity and cy topathogenicity.
42 citations
TL;DR: It is proposed that NNIs binding at three of the four distinct sites specifically inhibit the initiation step by the same mechanism: they prevent NS5B's "thumb" from quite reaching the proper initiation-competent position.
42 citations
TL;DR: 2 series of indole-N-acetamides are reported, bearing physicochemically diverse carboxylic acid replacements, which show potent affinity for the NS5B enzyme with reduced potential for formation of glucuronide conjugates, and preliminary optimization of these series furnished compounds that are potent in the blockade of subgenomic HCV RNA replication in HUH-7 cells.
41 citations