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

Abstract

Hepatitis C virus RNA-dependent RNA polymerase (ns5B polymerase) is a major target of drug development in the search for an effective treatment of hepatitis C. Various nucleoside analogs (NA) and allosteric non-nucleoside inhibitors (NNI) have been shown to bind ns5B polymerase and inhibit the replication of HCV. NNIs have been classified based on the drug binding site as observed by crystallography (Thumb I, Thumb II, Palm III, and Palm IV). However, the inherent variability of HCV and the rapid emergence of resistance mutations have highlighted the need for a better understanding of the mechanism of inhibition of NNIs at a molecular level. For that purpose, we have used Hydrogen-Deuterium exchange coupled to mass spectrometry to characterize the dynamics of ns5B polymerase in the presence and absence of an allosteric thumb II site NNI. As expected, we have determined that inhibitor binding to ns5B polymerase causes a significant loss of exchange at the binding site and in regions directly adjacent. Furthermore, we have observed that large regions of ns5B which are distant from the drug binding site display a significant loss in protein dynamics as detected by HD exchange. The observed network of allosterically suppressed dynamics encompasses the fingers, fingers extensions, thumb, C-terminal tail and beta-loop regions which have all been implicated in one of the multiple steps of the reaction cycle (de novo initiation, transition to elongation and processive elongation).