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

固有无序蛋白(IDPs)在阿尔茨海默氏症、乳腺癌以及丙型肝炎病毒和SARS-CoV-2等病毒中发挥着重要作用。因此，靶向障碍提供了巨大的治疗机会。然而，由于国内流离失所者的高度动态性质和缺乏明确的结构，他们往往被认为是无法下药的。非结构蛋白5A(NS5A)是一种来自丙型肝炎病毒的多功能磷酸蛋白，丙型肝炎病毒感染全球超过5800万人，并导致慢性肝病。NS5A的结构域I是高度结构化的，专门参与基因组复制，而结构域II和III是非结构化的，是产生传染性病毒颗粒所必需的。临床上广泛使用的现有疗法包括被认为针对NS5A的直接作用抗病毒药物(DAA)。这些药物非常有效，表现出皮摩尔活性，但它们的确切作用机制尚不清楚。汉森教授实验室进行的初步核磁共振(NMR)实验表明，其中一种药物Ledipasvir与NS5A的非结构化区域相互作用。这个博士项目旨在揭示NS5A抑制的细胞和生物物理机制。我的目标是了解现有的治疗方法是否针对NS5A的结构域和/或非结构域，以及这些相互作用是否导致抑制丙型肝炎病毒复制。在Towers实验室，将使用丙型肝炎病毒复制子试验来测试Ledipasvir和其他DAA对丙型肝炎病毒复制的作用，并确定NS5A无序和结构化区域对药物活性的重要性。在汉森实验室进行的核磁共振实验将被用来表征这些药物与NS5A的不同结构域的结合。这项研究将加深对NS5A靶向DAA治疗活性的理解，并在治疗丙型肝炎和针对其他蛋白质的药物设计中都具有重要意义，特别是那些具有无序区域的蛋白质。

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.