Structural and Functional Characterisation of Human Lysosomal Glycoside Hydrolases: Insights into Lysosomal Storage Disorders

Carbohydrates are one of the most abundant and diverse class of biomolecules on earth, and their importance in life is reflected by the range of carbohydrate active enzymes (CAZymes) which have evolved to process glycans in nature. Unsurprisingly, defects in these CAZymes have been linked to a range of human diseases. Of note, lysosomal storage disorders (LSDs) are a group of inherited metabolic disorders which result from deficiencies in particular lysosomal glycoside hydrolases. Understanding the enzymes which underpin these disorders is crucial to the development of effective diagnostic and therapeutic strategies. In this regard, the work described here sought to aid in the development of novel activity-based probes (ABPs), inhibitors and molecular chaperones for GBA and α-GAL, the enzymes associated with the LSDs Gaucher Disease (GD) and Fabry Disease (FD) respectively, to support the study of these enzymes in disease pathogenesis, diagnosis and treatment. In Chapter 2, the first ever co-crystal structures of human α-GAL in complex with several galacto-configured cyclophellitol-based inhibitors and ABPs for FD are described, providing key mechanistic and structural insight into their binding mode, reactivity and conformation. This work also expands to the characterisation of a non-covalent galacto-cyclophellitol cyclosulfamidate inhibitor, which exhibits chaperoning behaviour towards α-GAL as a potential pharmacological chaperone for the treatment of FD. In a parallel vein, the work presented in Chapter 3 describes a series of crystal structures in which human GBA is complexed with a variety of cyclophellitol ABPs and inhibitors for GD. Indeed, these co-crystal complexes enabled a structure-guided approach to the development of more selective GBA inhibitors, which proved suitable for the generation of neuropathic GD animal models through chemical knockdown. To support such work, Chapter 4 outlines the development of an insect-baculovirus expression vector system for the production of non-clinical human GBA. This enzyme has been a long-standing academic target which previously has only been reliably produced in industrial labs by unknown and irreproducible means. Lastly, using this recombinant GBA formulation, Chapter 5 discusses the structural analysis of a novel class of allylic carbasugar inhibitors for GBA, revealing multiple covalent and non-covalent mechanisms which may provide structural bases for their stabilising behaviour as potential pharmacological chaperones for GD. It is ultimately hoped that this work will support future developments in the field of LSD research, particularly with regard to the diagnosis and treatment of such disorders.