Characterization of ketohexokinase as a therapeutic target for hereditary fructose intolerance and metabolic syndrome

Abstract

Over the past forty years, there has been an increase in obesity, diabetes, and heart disease, collectively known as metabolic syndrome (MetS), in which fructose has been implicated. In addition to MetS, hereditary fructose intolerance (HFI) has no known treatment aside from the difficult removal of fructose from the diet. Ketohexokinase (KHK) is the first enzyme in the fructose metabolic pathway and catalyzes an ATP-dependent reaction that phosphorylates fructose to fructose 1-phosphate. For effective inhibitor development, it is key to understand the KHK-catalytic mechanism. To that end, the research described in this thesis focuses on two goals: 1) understanding how KHK functions in its role as a metabolic enzyme, using structure-function analysis to inform the development of KHK inhibitors, and 2) investigating how these findings can be used to make KHK a prime therapeutic target for alleviating diseases such as HFI and MetS. The X-ray crystal structure of the mouse-liver isozyme, KHK-C (mKHK-C), was determined at a resolution of 1.79 Å. The mKHK-C structure is in complex with the substrate fructose and the product of catalysis, ADP, forming a ground-state complex. The mKHK-C structure has nearly identical secondary structure to its human homolog and has similar steady-state kinetic parameters validating the use of mouse models for exploring the pre-clinical efficacy of KHK-C inhibitors. Furthermore, six structures of human KHK-C in complex with inhibitors and ligands are presented. These structures support the kinetic analyses showing these inhibitors are all competitive with ATP and reveal the shape and polarity of the ATP-binding pocket to achieve inhibition constants (Ki) as low as 50 nM. Lastly, comparison of all KHK structures demonstrate that the β-sheet domain of KHK is capable of 30.3° rotation of the β-sheet domain towards the active site of the opposing dimer subunit. Kinetic experiments using site-directed mutants of human KHK-C and various viscogens confirmed that a conformational change is linked to KHK’s catalytic function. This research provides a foundation for further development of more specific KHK inhibitors aimed at HFI and MetS therapies.