Use this URL to cite or link to this record in EThOS:
Title: Towards a structural understanding of the mechanism of action of dimeric HCV inhibitors
Author: Lambert, Sebastian Mark
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
Availability of Full Text:
Access from EThOS:
Access from Institution:
Hepatitis C Virus (HCV) is expected to be an increasing global healthcare burden. HCV Infection can progress to major chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). No protective vaccine currently exists against hepatitis C and current therapies are ineffective. NS5A is an essential component of the HCV with roles in RNA replication, modulation of the host cell and assembly of viral particles, however no precise function of NS5A has been established. New direct acting antivirals (DAA) such as daclatasvir (DCV), a dimeric compound which targets NS5A with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however resistance mutations appear in the NS5A domain 1. Genotype 1a HCV is less affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A genotype and intriguingly, our X-ray crystal structure shows two new dimeric forms of this domain. Our new NS5A domain 1 dimers serve as additional candidate targets for inhibitors and could explain the role of NS5A in the regulation of HCV replication. The high solvent content (75%) of our crystal form makes it ideal for ligand-soaking, however we did not observe the presence of DCV in soaks with our crystals. Using nuclear magnetic resonance (NMR) spectroscopy, we further investigated the binding of DCV to NS5A constructs which included the key resistance mutation sites. These extended constructs appeared to have slightly altered conformations which allow a weak interaction with DCV. This data and subsequent studies will give new insights into the roles of NS5A in the HCV replication complex and the mechanism of action of this class of DAA.
Supervisor: Matthews, Stephen Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral