Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.

Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in β-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activitybased probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N-functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA's preference for O6-and N-functionalised reagents, a bi-functional aziridine ABP was synthesized as a potentially more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.

Deficiency in human acid glucosylceramidase (GBA1, a retaining β‐glucosidase) causes the lysosomal sphingolipid storage disorder Gaucher disease, whereas deficiency in human acid α‐glucosidase (GAA, a retaining α‐glucosidase) triggers the lysosomal glycogen storage disorder Pompe disease. Both enzymes process their substrate following a two‐step double‐displacement mechanism involving a covalent enzyme–substrate intermediate. Structural analysis of glycosidases complexed to substrates and inhibitors has provided insight into the reaction coordinates followed by glycosidases during catalytic hydrolysis and has assisted in the design of potent and selective inhibitors. Competitive and covalent inhibitors of both GBA1 and GAA have been developed in the past decades, for fundamental studies, as diagnostics tools, leads for drug development and therapeutic drugs for the clinical treatment of lysosomal storage diseases. Key Concepts The structural and functional diversity of glycoconjugates is reflected by the vast number of glycoprocessing enzymes encountered in all domains of life. Retaining glycosidases form a covalent intermediate during substrate processing, whereas inverting glycosidases do not. Competitive and covalent retaining glycosidase inhibitors exist, whereas inverting glycosidases can normally only be blocked by competitive inhibitors. Both covalent and competitive glycosidase inhibitors are found in nature and serve as inspiration for the design of synthetic inhibitors. Structural analysis of glycosidases complexed to substrates and inhibitors provides insight into the reaction coordinates followed by glycosidases during the catalytic cycle. Conformational analysis of the reaction coordinate of glycosidases is key in the design of potent and selective inhibitors. Glycosidase inhibitors may serve as leads for drug development. Activity‐based glycosidase probes can be applied in the discovery of glycosidase activities. Activity‐based probes are useful starting points for the development of diagnostics assays in disease areas in which glycosidase activities are involved. Genetic deficiency in glycosidases is the basis of numerous inherited disorders including Gaucher disease and Pompe disease.

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