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Identification and Biological Characterization of Heterocyclic Inhibitors of the Hepatitis C Virus RNA-dependent RNA Polymerase
Journal article   Open access   Peer reviewed

Identification and Biological Characterization of Heterocyclic Inhibitors of the Hepatitis C Virus RNA-dependent RNA Polymerase

Dashyant Dhanak, Kevin J. Duffy, Victor K. Johnston, Juili Lin-Goerke, Michael Darcy, Antony N. Shaw, Baohua Gu, Carol Silverman, Adam T. Gates, Michael R. Nonnemacher, …
The Journal of biological chemistry, v 277(41), pp 38322-38327
11 Oct 2002
PMID: 12167642
url
http://www.jbc.org/content/277/41/38322.full.pdfView
Published, Version of Record (VoR) Open
url
https://doi.org/10.1074/jbc.M205566200View
Published, Version of Record (VoR) Open

Abstract

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of anin vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC50 of ∼0.5 μm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.

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Biochemistry & Molecular Biology
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