HCV Replicon Inhibitors: Mode of Action and Resistance Mapping
The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, we reported the identification of benzo-1,2,4-thiadiazine derivatives as potent heterocyclic inhibitors of the HCV RdRp. These agents interact directly with the viral polymerase and have been shown to be highly specific for HCV. Compounds 1 and 4 reduced viral RNA in replicon cells with an IC5o around 500 nM and have a mode of action distinct from interferon-alpha (IFN-a) or ribavirin based on drug combination studies. Furthermore, complete and rapid inhibition of HCV replicon RNA synthesis was apparent in a time course analysis using 54, resulting in a loss of viral RNA consistent with replicon RNA half-life. Maximal inhibition of replication was reached prior to 50 h post-treatment with compound 4, and this inhibition of viral RNA synthesis was sustained through 78 h post-treatment. A similar dose-response inhibition profile, although with reduced kinetics, was apparent when treating replicon cells with a concentration of compound 4 approximating the replicon IC50 or with ribavirin. In contrast, the kinetic profile for inhibition of viral RNA synthesis after IFN-a treatment was distinct, showing (a) a delay of approximately 4 h prior to inhibition of replication, (b) a transient response, whereby inhibition of RNA synthesis by IFN-a treatment was sustained for only the first 50 h, followed by a rapid increase in replication and, (c) a threshold amount of IFN-a was required to initiate an antiviral state, as measured by induction of human p68 message, resulting in the subsequent reduction of replicon RNA. Lastly, selection of antiviral resistance for the benzothiadiazine inhibitors in the replicon system will be discussed.
KeywordsViral Polymerase Inhibition Profile Replicon Cell NS5B Protein Replicon System
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