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

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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.