Cellular and biophysical mechanisms of small-molecule inhibition of non-structural protein 5A.

Intrinsically disordered proteins (IDPs) play important roles in diseases such as Alzheimer's, breast cancer, and viruses, including Hepatitis C virus (HCV) and SARS-CoV-2. As such, targeting disorder offers huge therapeutic opportunity. However, IDPs are often considered undruggable due to their highly dynamic nature and the absence of well-defined structure. Non-structural protein 5A (NS5A) is a multi-functional phosphoprotein from HCV, a virus which affects over 58 million people worldwide and causes chronic liver disease. Domain I of NS5A is highly structured and exclusively involved in genome replication, while domains II and III are unstructured and required for the production of infectious viral particles. Existing therapies in widespread clinical use comprise of direct acting antivirals (DAAs) which are believed to target NS5A. These drugs are exceptionally potent, exhibiting picomolar activities, however their exact mechanisms of action remain unknown. Preliminary nuclear magnetic resonance (NMR) experiments undertaken in Professor Hansen's lab show that one such drug, ledipasvir, interacts with the unstructured region of NS5A.
This PhD project aims to uncover the cellular and biophysical mechanisms of NS5A inhibition. I aim to understand whether existing therapeutics target the structured and/or unstructured domains of NS5A, and if these interactions result in inhibition of HCV replication. In the Towers lab, an HCV replicon assay will be employed to test ledipasvir and other DAAs against HCV replication and determine the significance of the disordered and structured regions of NS5A on the activity of the drugs. NMR experiments, undertaken in the Hansen lab, will be used to characterise the binding of these drugs to various domains of NS5A. This research will deepen understanding of the therapeutic activity of NS5A-targeting DAAs and be important both in the treatment of HCV, and in the design of drugs targeting other proteins, particularly those with disordered regions.