ALLOSTERIC INHIBITION IN VIRAL POLYMERASES: UNDEERSTANDING THE IMPACT OF MULTIPLE INHIBITORS AND PREDICTING NOVEL BINDING SITES USING COMPUTATIONAL CHEMISTRY APPROACHES

Abstract

Viral pathogens are responsible for a large number of human infections. For example, the Hepatitis C virus (HCV) affects close to 3% of the world?s population. There has been significant progress in the therapeutic options available for the treatment of HCV and most of the highly effective existing therapies are a combination of small molecule inhibitors targeting key viral enzymes. One such target is the polymerase, which is a critical component of the viral life cycle. Our work focuses on understanding inhibition of the polymerase by small molecules that bind to pockets outside the active site (i.e. allosteric inhibitors). The first objective of this study is to determine how allosteric inhibitors alter the structural, dynamic and thermodynamic properties of the HCV polymerase so that these small molecules can synergistically inhibit the enzyme. Our second goal is to identify putative allosteric sites in the polymerases of the Dengue (DENV), West Nile (WNV) and Foot-and-mouth Disease (FMDV) viruses. One of the existing challenges in current HCV treatment is that low fidelity of NS5B increases the rate of mutations in the virus. This results in the generation of multiple enzyme variants and makes it difficult to target the enzyme with single inhibitors. In contrast, for DENV, WNV and FMDV one of the major hurdles is lack of biochemical and structural data that can help steer more structure-based drug discovery efforts. Results from our studies of the HCV polymerase suggest that allosteric inhibitors from nonoverlapping sites can bind simultaneously and synergistically modulate the enzyme conformation and free energy landscape. This work provides the first molecular descriptions of mechanisms underlying enhanced inhibition of the HCV polymerase. We used validated HCV allosteric sites as targets to evaluate several ligand binding site predictor tools and used the best tool to identify novel putative allosteric pockets in the DENV, WNV and FMDV polymerases. The identified sites displayed structural and/or chemical similarities with two of the NS5B validated allosteric pockets. Our approach provides an economic and faster means to facilitate an essential step of drug discovery (i.e. binding site identification), which is critical for infections with limited therapeutic options.