Activity‐based glycosidase profiling: monitoring glycosphingolipid metabolism in health and disease

Activity‐based protein profiling allow the discovery and monitoring of active enzymes in their biological surroundings. An activity‐based probe (ABP) consists of a substrate analogue featuring a reactive group that binds to the enzyme (or enzyme family) of interest in a covalent and irreversible manner, as well as a reporter moiety, which can be an affinity tag, a fluorophore or a bioorthogonal tag. Enzyme families that form a covalent bond in their substrate processing are most suited for ABP design and therefore most ABP applications in the past decade have focused on ABPs for serene hydrolyses, cysteine proteases and threonine proteases. Retaining glycosidases employ a Koshland two‐step double displacement in the hydrolysis of glycosidic bonds. During the catalytic cycle a covalent bond with the substrate glycoside is formed along with expulsion of the aglycon leaving group. The natural product, cyclophellitol, is a potent mechanism‐based retaining beta‐glucosidase inhibitor that capitalises on this mechanism by forming a stable, covalent enzyme‐inhibitor adduct. Grafting a reporter molecule on cyclophellitol or its nitrogen analogue, cyclophellitol aziridine, delivers a potent and selective retaining beta‐glucosidase ABP. Variation of the cyclophellitol/cyclophellitol aziridine configuration yields ABPs selective for a variety of retaining gluycosidases including lysosomal galactocerebrosidase, lysosomal and ER alpha glucosidase and a number of retaining beta‐glucuronidases. In this contribution I will present our work on the design synthesis and application of a broad panel of retaining glycosidase ABPs. I will focus on the application of these tools in the functioning of the underlying glycosidases in health and disease, with special attention to glycosidases involved in lysosomal storage disorders. Support or Funding Information ERC Advanced Grand ‘CHEMBIOSPHING’ Netherlands Organization for Scientific Research