Small molecule tools to target glucose metabolism in Non-Alcoholic Fatty Liver Disease (NAFLD)

非酒精性脂肪性肝病（NAFLD）是包括肝脂肪变性、非酒精性脂肪性肝炎（NASH）、肝纤维化、肝硬化和肝细胞癌（HCC）在内的一系列疾病。NAFLD目前是慢性肝病的主要病因，在全世界的患病率为25%。尽管NAFLD是一个全球性的卫生保健问题，但对NAFLD的总体认识仍然非常有限，因此目前没有治疗或预防的治疗方法。在世界范围内，NAFLD的患病率正随着2型糖尿病和肥胖症的增加而成比例地增加。此外，观察到NAFLD导致的终末期肝病患者数量迅速增加，从2004-2013年（美国），肝移植等待名单上的NASH患者增加了170%。因此，越来越明显的是，需要进一步研究可用于解决这一日益严重的公共卫生问题的非侵入性治疗方法和工具。NALFD是目前肝病研究中发展最快的领域，预计在未来十年内，可用的治疗方法将发生巨大的转变。本项目的主要目的和目标是设计和合成靶向NAFLD中葡萄糖代谢的小分子工具。我们的分子靶向葡萄糖代谢，因为我们假设肝脂肪变性和随后的脂肪变性进展到NASH和纤维化是由肝脏高葡萄糖处置引起的。这分别是饮食中碳水化合物过量和高胰岛素血症或基因变异引起的葡萄糖激酶表达或活性升高的结果。因此，我们建议下调肝脏葡萄糖激酶（GK）蛋白或直接抑制酶可能导致肝脏葡萄糖清除率降低。GK活性的降低可以通过选择性抑制或选择性蛋白质降解来实现，从而限制肝脏葡萄糖清除，并可能为NAFLD提供有效的治疗方法。这个项目有两个方面，将并行处理。主要重点是GK抑制剂的合成，其作用是促进GK与其调节蛋白GKRP的结合。这些工具化合物的开发将通过确定小分子是否可抑制GK，提供宝贵的概念验证研究。据我们所知，用小分子抑制葡萄糖激酶还没有报道。该项目的这方面是与阿斯利康合作的，阿斯利康已经进行了高通量筛选（HTS），以确定可能抑制GK的潜在小分子。HTS已经收到，三个热门系列已经在纽卡斯尔大学进一步验证。将测试化合物的GK活性，并根据它们与GK和GKRP的相互作用进行优化。稳健的合成路线将被开发，以建立一个化合物库和启动hit-to-lead研究。此外，本项目将通过靶向嵌合体（Proteolysis Targeting Chimeras, PROTACs）研究GK的降解。GK激活剂（GKA’s）在短期试验中成功地降低了血糖浓度，因此此前已被开发为2型糖尿病的可能治疗策略。然而，在长期的临床研究中，许多GKA的疗效消失了。利用这些成熟的GKA支架，我们提出可以合成GK PROTACs。PROTAC将由一个E3连接酶的配体与一个合适的连接体连接到GK配体上。由于已经建立的合成路线和对GK的高亲和力，GKA将作为GK的有效配体。第二代PROTACs将通过用GK抑制剂（GKI）取代GKA组分合成，并将降解GK和GKRP。

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of diseases including hepatic steatosis, non-alcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). NAFLD is now the leading cause of chronic liver disease and has a 25% prevalence worldwide. Despite being a global healthcare problem, the overall understanding of NAFLD remains very limited and as such there are no therapeutics currently available for treatment or prevention. The prevalence of NAFLD is increasing proportionately with the increase of type 2 diabetes mellitus and obesity worldwide. Additionally, a rapid increase in the number of patients with final stage liver disease as a result of NAFLD, has been observed and from 2004-2013 (USA) there was a 170% increase in NASH patients on the liver transplant waiting list. It has therefore become increasingly apparent that further research is needed into non-invasive therapeutics and tools that can be used to combat this ever-growing public health problem. NALFD is currently the fastest growing area of liver disease research and it is predicted that a huge transformation in the available therapeutics will occur over the next decade.The key aims and objectives of this project are the design and synthesis of small molecule tools that target glucose metabolism in NAFLD. Our molecules target glucose metabolism as, we hypothesise that hepatic steatosis and the subsequent progression of steatosis through to NASH and fibrosis is caused by high hepatic glucose disposal. This is a result of dietary carbohydrate excess and elevated glucokinase expression or activity caused as a result of hyperinsulinaemia or genetic variants, respectively. We are therefore proposing that downregulation of liver glucokinase (GK) protein or direct enzyme inhibition could result in decreased hepatic glucose clearance. This lowering of GK activity could be achieved by selective inhibition or selective protein degradation leading to restricted hepatic glucose clearance and may provide an effective therapeutic for NAFLD. This project has two strands that will be approached in parallel. The primary focus is the synthesis of GK inhibitors which work by promoting binding of GK to its regulatory protein, GKRP. Development of these tool compounds will provide an invaluable proof-of-concept study by establishing whether GK inhibition is tractable with a small molecule. To our knowledge, inhibition of glucokinase with a small molecule has not been reported. This aspect of the project is in collaboration with AstraZeneca, where a High Throughput Screen (HTS) has been performed in order to identify potential small molecule hits that could inhibit GK. The HTS has been received and three hit series have been further validated here at Newcastle University. Compounds will be tested for GK activity and will be optimised based on their interactions with both GK and GKRP. Robust synthetic routes will be developed in order to build a library of compounds and initiate hit-to-lead studies. Additionally, this project will explore degradation of GK by using Proteolysis Targeting Chimeras (PROTACs). GK Activators (GKA's) have previously been developed as a possible therapeutic strategy for type 2 diabetes, as they were successful in lowering blood glucose concentration in short term trials. However, the efficacy of many GKA's was lost during long-term clinical studies. Using these well-established GKA scaffolds we propose that GK PROTACs can be synthesised. The PROTAC would consist of a ligand for an E3 ligase attached to the GK ligand with an appropriate linker. With an already established synthetic route and high affinity for GK, the GKA's will provide as an effective ligand for GK. A second generation of PROTACs will be synthesised by replacing the GKA component with a GK inhibitor (GKI) and will degrade both GK and GKRP.