Browsing by Author "Davis, Brittny C."
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Item Allosteric Inhibitors Have Distinct Effects, but Also Common Modes of Action, in the HCV Polymerase(2016-11-15) Davis, Brittny C.; Brown, Jodian A.; Thorpe, Ian F.The RNA-dependent RNA polymerase from the Hepatitis C Virus (gene product NS5B) is a validated drug target because of its critical role in genome replication. There are at least four distinct allosteric sites on the polymerase to which several small molecule inhibitors bind. In addition, numerous crystal structures have been solved with different allosteric inhibitors bound to the polymerase. However, the molecular mechanisms by which these small molecules inhibit the enzyme have not been fully elucidated. There is evidence that allosteric inhibitors alter the intrinsic motions and distribution of conformations sampled by the enzyme. In this study we use molecular dynamics simulations to understand the structural and dynamic changes that result when inhibitors are bound at three different allosteric binding sites on the enzyme. We observe that ligand binding at each site alters the structure and dynamics of NS5B in a distinct manner. Nonetheless, our studies also highlight commonalities in the mechanisms of action of the different inhibitors. Each inhibitor alters the conformational states sampled by the enzyme, either by rigidifying the enzyme and preventing transitions between functional conformational states or by destabilizing the enzyme and preventing functionally relevant conformations from being adequately sampled. By illuminating the molecular mechanisms of allosteric inhibition, these studies delineate the intrinsic functional properties of the enzyme and pave the way for designing novel and more effective polymerase inhibitors. This information may also be important to understand how allosteric regulation occurs in related viral polymerases and other enzymes.Item Thumb inhibitor binding eliminates functionally important dynamics in the hepatitis C virus RNA polymerase(Wiley, 2012-07-31) Davis, Brittny C.; Thorpe, Ian F.Hepatitis C virus (HCV) has infected almost 200 million people worldwide, typically causing chronic liver damage and severe complications such as liver failure. Currently, there are few approved treatments for viral infection. Thus, the HCV RNA‐dependent RNA polymerase (gene product NS5B) has emerged as an important target for small molecule therapeutics. Potential therapeutic agents include allosteric inhibitors that bind distal to the enzyme active site. While their mechanism of action is not conclusively known, it has been suggested that certain inhibitors prevent a conformational change in NS5B that is crucial for RNA replication. To gain insight into the molecular origin of long‐range allosteric inhibition of NS5B, we employed molecular dynamics simulations of the enzyme with and without an inhibitor bound to the thumb domain. These studies indicate that the presence of an inhibitor in the thumb domain alters both the structure and internal motions of NS5B. Principal components analysis identified motions that are severely attenuated by inhibitor binding. These motions may have functional relevance by facilitating interactions between NS5B and RNA template or nascent RNA duplex, with presence of the ligand leading to enzyme conformations with narrower and thus less accessible RNA binding channels. This study provides the first evidence for a mechanistic basis of allosteric inhibition in NS5B. Moreover, we present evidence that allosteric inhibition of NS5B results from intrinsic features of the enzyme free energy landscape, suggesting a common mechanism for the action of diverse allosteric ligands. Proteins 2013. © 2012 Wiley Periodicals, Inc.