Cyclophellitol

α-Glucosidase inhibitors are potential therapeutics for the treatment of diabetes, viral infections, and Pompe disease. Herein, we report a 1,6-epi-cyclophellitol cyclosulfamidate as a new class of reversible α-glucosidase inhibitors that displays enzyme inhibitory activity by virtue of its conformational mimicry of the substrate when bound in the Michaelis complex. The α-d-glc-configured cyclophellitol cyclosulfamidate 4 binds in a competitive manner the human lysosomal acid α-glucosidase (GAA), ER α-glucosidases, and, at higher concentrations, intestinal α-glucosidases, displaying an excellent selectivity over the human β-glucosidases GBA and GBA2 and glucosylceramide synthase (GCS). Cyclosulfamidate 4 stabilizes recombinant human GAA (rhGAA, alglucosidase alfa, Myozyme) in cell medium and plasma and facilitates enzyme trafficking to lysosomes. It stabilizes rhGAA more effectively than existing small-molecule chaperones and does so in vitro, in cellulo, and in vivo in zebrafish, thus representing a promising therapeutic alternative to Miglustat for Pompe disease.

The ability to detect active enzymes in a complex mixture of folded proteins (e.g., secretome, cell lysate) generally relies on observations of catalytic ability, necessitating the development of an activity assay that is compatible with the sample and selective for the enzyme(s) of interest. Deconvolution of the contributions of different enzymes to an observed catalytic ability further necessitates an often-challenging protein separation. The advent of broadly reactive activity-based probes (ABPs) for retaining glycoside hydrolases (GHs) has enabled an alternative, often complementary, assay for active GHs. Using activity-based protein profiling (ABPP) techniques, many retaining glycoside hydrolases can be separated, detected, and identified with high sensitivity and selectivity. This chapter outlines ABPP methods for the detection and identification of retaining glycoside hydrolases from microbial sources, including protein sample preparation from bacterial lysates and fungal secretomes, enzyme labeling and detection via fluorescence, and enzyme identification using affinity-based enrichment coupled to peptide sequencing following isobaric labeling.

Exo-β-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-β-mannosidases in biological and biomedical research.