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Molecular mechanism of highly potent NS5A inhibitors

Navarro Garcia, Elizabeth Melissa 2019. Molecular mechanism of highly potent NS5A inhibitors. PhD Thesis, Cardiff University.
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Abstract

Hepatitis C is responsible for causing chronic infections in over 170 million people all over the world who are at a risk of developing into liver cirrhosis and hepatocellular carcinoma, locating HCV in a major public health burden. Until recently, the standard-of care treatment consisted of Interferon-alpha and ribavirin, in addition to non-structural protein 3/4 (NS3) protease inhibitors, but due to the undesired side-effects, researchers developed more efficient therapies. Nowadays, small molecules targeting non-structural viral proteins: NS3/4 protease, NS5A D1 and NS5B polymerase activities can clear the infection in 98% of the cases. These direct acting antivirals (DAAs) are widely used, however, despite advances in recently approved potent DAAs the world-wide application of these therapies remains limited due to the expensive cost and potential drug resistance. NS5A is a nonstructural multifunctional protein. Mainly composed by an amphipatic helix, which is the major membrane anchor, Domain I, which is involved in RNA binding and assembly, and Domain II and III which are intrinsically unfolded domains and are known to interact with host factors. DAA targeting NS5A DI, Daclatasvir (DCV), has a picomolar range activity and it is used in combination therapy to combat HCV infection. Given the enormous medical relevance of NS5A inhibitors, the aim of this study was to decipher the mode of action of Daclatasvir, together with more insights to the role of NS5A structural elements. In the present study, experiments showed that DCV can block the envelopment of viral particles. Furthermore, targeting the assembly of HCV particles, this fact serve as evidence of the dual mode of action of DCV. Furthermore, we investigated the role of very conserved Proline residues in the structure of NS5A, identifying key Proline residues which are critically involved in RNA replication, and have an impact in HCV infection. This fact, also suggests that the some of these Prolines might be essential for the DCV binding, as we prove that they have a direct role in keeping the binding site of DCV. Lastly, we set up a molecular model which includes the intracellular membrane giving the full picture of how DCV works in the context of an intracellular membrane and its important interactions. Together our data, prove the dual mode of action of DCV targeting HCV replication and assembly. And importantly, we constructed a molecular model that can be use in the future to study structure-function of developing NS5A inhibitors.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Pharmacy
Subjects: R Medicine > RS Pharmacy and materia medica
Date of First Compliant Deposit: 14 June 2019
Last Modified: 14 Jun 2019 10:29
URI: http://orca-mwe.cf.ac.uk/id/eprint/123434

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