Glycosidases are enzymes that catalyze the cleavage of glycosidic bonds. Because of their roles in metabolism, protein post-translational modifications, cell-cell interactions, as well as in viral and bacterial infections, glycosidases have been the target of various therapeutic interventions. The development of glycosidase inhibitors has been useful for increasing knowledge of mechanistic details of their corresponding target enzymes and for probing the functions of specific glycoconjugates. This thesis describes a rapid method for discovering potent, selective inhibitors for □- fucosidase (Fuc) and N-acetyl-□-hexosaminidase (Hex). These inhibitors were either investigated for their binding interactions using X-ray crystallography, isothermal titration calorimetry, and pH profile analysis, or were evaluated at the cellular level to explore their potential for use in future applications. Chapter 1 describes the significance of, and presents background information on, glycosidases and their inhibitors.
We developed an efficient method for examining the activity and selectivity of various inhibitors on two □-fucosidases - one (TmF) from Thermotoga maritima and the other (HuF) from human in this study. A variety of fuconojirimycin (FNJ) derivatives with substitution at C1, C2, C6, or N were prepared in microplates and then screened without purification to assess their inhibitory activity levels on the two □-fucosidases. Among the FNJ derivatives tested, the majority of the C1-substituted FNJs were slow, tight-binding inhibitors of TmF, but acted as reversible inhibitors of HuF. The best C1-substituted inhibitor exhibited a 13,700-fold difference in affinity between the two □-fucosidases (Chapter 2).
We applied C1-substituted FNJ derivatives to distinguish between TmF and Corynebacterium fucosidase. The flexibility of the loop (TmF sequence 44-65) was found to be critically associated with inhibition potency (Chapter 3). Subsequently, to determine the dynamic motion of the Fuc/inhibitor that interact from low to high binding affinities, we obtained nine X-ray structures, consisting of TmF and FNJ derivative complexes. The structures had dissociation constant (Ki) values in the □M to pM range. The analysis of these complex structures identified several factors important in improving binding affinity. The low □M Ki level structures provided sufficient electrostatic and H-bond interactions for stabilization of loops 1 and 2, in the main control of Y64, D224 and E266. Further improvement of Ki from the nM to pM is attributed to the increase of hydrophobic interactions and entropy. The flexibility of the aglycone portion and the resulting hydrophobic and H-bond interactions likely contributed to further fine-tuning of affinities in the Ki pM range (Chapter 4).
Previous works have provided evidence of the uptake of L-fucose from gastric cancer cells to H. pylori. In that study, fucosidase activity was detected in the culture medium of H. pylori-infected gastric cancer cells. Here, for the purpose of rapid and efficient purification, the previously developed 1-aminomethyl-1-deoxy-FNJ was immobilized to agarose beads. The resulting affinity chromatography and subsequent liquid chromatography-tandem mass spectrometric analysis identified human □-L-fucosidases 2 as the key enzyme involved in the aforementioned L-fucose transfer (Chapter 5).
Finally, human Hex isozymes are considered important glycosidase targets for drug discovery because of their connection to osteoarthritis and lysosomal storage disorders. We developed GlcNAc-type iminocyclitols as potent and selective Hex inhibitors. The most potent inhibitor had a Ki of 0.69 nM against human Hex B and was 2.5□105 times more selective for Hex B than for a similar human enzyme, O-GlcNAcase. These glycosidase inhibitors were shown to modulate intracellular levels of glycolipids, including ganglioside-GM2 and asialoganglioside-GM2 (Chapter 6).

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