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

非酒精性脂肪性肝病（NAFLD）是一系列疾病，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）。NAFLD现在是慢性肝病的主要原因，在全球范围内的患病率为25%。尽管NAFLD是一个全球性的医疗保健问题，但对NAFLD的总体了解仍然非常有限，因此目前没有可用于治疗或预防的治疗方法。随着2型糖尿病和肥胖的增加，NAFLD的患病率也在增加。此外，已观察到由于NAFLD而患有终末期肝病的患者数量迅速增加，并且从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，需要进一步研究非侵入性疗法和工具，以应对这一日益严重的公共卫生问题。NALFD是目前肝病研究中发展最快的领域，预计未来十年将出现可用治疗方法的巨大转变。该项目的主要目的和目标是设计和合成针对NAFLD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为NASH和纤维化是由高肝葡萄糖处置引起的。这是饮食碳水化合物过量和葡萄糖激酶表达或活性升高的结果，分别由高胰岛素血症或遗传变异引起。因此，我们提出下调肝脏葡萄糖激酶（GK）蛋白或直接抑制酶可导致肝脏葡萄糖清除率降低。这种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，从而导致肝脏葡萄糖清除受限，并且可以为NAFLD提供有效的治疗。该项目有两个方面，将平行进行。主要焦点是GK抑制剂的合成，其通过促进GK与其调节蛋白GKRP的结合而起作用。这些工具化合物的开发将提供一个宝贵的概念验证研究，通过确定GK抑制是否是易于处理的小分子。据我们所知，用小分子抑制葡萄糖激酶还没有报道。该项目的这一方面是与阿斯利康合作，在那里进行了高通量筛选（HTS），以确定可能抑制GK的潜在小分子命中。HTS已经收到，三个命中系列已在纽卡斯尔大学进一步验证。将测试化合物的GK活性，并基于其与GK和GKRP的相互作用进行优化。将开发稳健的合成路线，以建立化合物库，并启动命中铅研究。此外，该项目将探索通过使用蛋白水解靶向嵌合体（PROTAC）降解GK。GK激活剂（GKA）以前已被开发为2型糖尿病的可能治疗策略，因为它们在短期试验中成功降低血糖浓度。然而，在长期临床研究中，许多GKA的疗效丧失。使用这些完善的GKA支架，我们建议可以合成GK PROTAC。PROTAC将由E3连接酶的配体组成，该配体通过适当的接头连接到GK配体。由于已经建立的合成路线和对GK的高亲和力，GKA将作为GK的有效配体提供。第二代PROTAC将通过用GK抑制剂（GKI）替代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.