Thumb II Site Inhibitor Allosterically Suppresses the Dynamics of HCV NS5B RNA‐Dependent RNA Polymerase

Abstract

The NS5B RNA‐dependent RNA polymerase of human hepatitis C virus (HCV) is a major target for drug development in the search for an effective treatment of hepatitis C. Various nucleoside analogs and allosteric non‐nucleoside inhibitors, classified based on the drug binding site, have been shown to bind NS5B and inhibit HCV replication. However, the genetic diversity of HCV and the rapid development of resistance mutations have stressed the need for better understanding of the mechanism of inhibition at a molecular level. With this intention, we have used hydrogen/deuterium exchange coupled with mass spectrometry to characterize the local dynamics of NS5B in the presence and absence of these inhibitors. We have verified that the inhibitor binding to the polymerase causes significant loss of exchange at the binding site and in regions adjacent. Additionally, we have seen that large regions of NS5B that are distant from the drug‐binding site show a significant loss in protein dynamics as detected by H/D exchange. This allosteric inhibition encompasses specific regions of NS5B that are involved in its catalytic cycle, including the fingers, finger extensions and thumb.Funding Source: University of Maryland School of Pharmacy