Details

Autor(en) / Beteiligte

• Kulkarni, Anupriya Shrikant

Titel

Mechanisms of Action and Resistance to Novel Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2015

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The hepatitis C virus (HCV) is a positive-sense RNA virus that encodes a non-structural protein, NS5B, which is an RNA-dependent RNA polymerase. NS5B is required for replication of the viral genome, making it an attractive target for antiviral drug development efforts. NS5B inhibitors are classified into two major categories: nucleoside inhibitors (NIs) and non-nucleoside inhibitors (NNIs). NIs bind to the enzyme active site and compete with the natural NTP for incorporation. In contrast, NNIs bind away from the active site and inhibit the polymerase activity allosterically, specifically during initiation of RNA synthesis by introducing conformational changes in the polymerase. The NS5B polymerase lacks proofreading ability, which results in a high rate of mutations during replication. This error prone nature of the HCV polymerase has given rise to a number of variants of the virus which are classified into different genotypes and subgenotypes. Different HCV genotypes show variations in drug susceptibility, particularly to NNIs, which limits their clinical utility. At least four distinct binding sites for NNIs have been identified on the NS5B protein and these binding sites are not necessarily conserved across the genotypes. This provides a possible explanation for the observed variations in drug susceptibility. As part of the first study described in this thesis, we tested the inhibitory activity of acyl pyrrolidine and 1,5-benzodiazepine against purified NS5B enzymes that represent the major HCV genotypes. We identified natural amino acid substitutions that contribute to resistance to the NNIs. In the second study described herein, we turned our attention to the mechanisms of action and resistance to NIs, with focus on sofosbuvir, which is part of the current standard of care in anti-HCV therapy. Previous crystallographic data of HCV NS5B in complex with an RNA primer-template has provided important information on the protein-nucleic acid interface during the elongation process. The structure points to specific interactions between residues of the nucleic acid binding channel and the 2'-hydroxyl group of the bound RNA substrate. Using templates with strategically engineered DNA-like residues, we examined the role of the 2'-hydroxyl group of the template strand in nucleotide incorporation. Our biochemical findings suggest that this 2'-hydroxyl group plays an essential role in establishing resistance to NIs, and this process may be mediated by the signature S282T mutation, which is been known to cause resistance to sofosbuvir. In the third study, we further evaluated the efficacy of novel NIs against known NI resistance-conferring mutations in NS5B. The compounds 2'F-2'-C-Me-UTP (sofosbuvir), 2'-C-Me-2'-NH2-UTP, 2'-C-Me-UTP and 2'-C-Me-(1-Thio)UTP, which were modified derivatives of sofosbuvir, were tested against mutations S282T, L159F, C316N and L320F. It was observed that S282T showed resistance to all the modified compounds. Mutations L159F, C316N and L320F showed sensitivity towards 2'-C-Me-UTP. WT NS5B, L159F, C316N and L320F showed increased IC50 values towards 2'-C-Me-2'-NH2-UTP and 2'-C-Me-(1-Thio) UTP suggesting that addition of NH2 and Thio groups do not increase the efficacy of the compounds. Overall, these studies provide a more detailed understanding of mechanisms of action and resistance to Nucleoside and Non-nucleoside inhibitors of the HCV polymerase. Our findings offer new avenues in current efforts to develop new viral polymerase inhibitors.