New irreversible α-L-iduronidase inhibitors and activity-based probes.

Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination on L-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-L-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-L-iduronidase (rIDUA, Aldurazyme®). The structure of IDUA in complex with the inhibitors in a non-covalent transition state mimicking form and covalent enzyme-bound form provides insights into the conformational itinerary employed by IDUA. Inhibitors 1-3 adopt a half-chair conformation in solution (4H3 and 3H4), as observed by DFT calculations, which is different from the Michaelis complex conformation observed by crystallographic studies, and consequently 1-3 may need to overcome an energetic barrier in order to switch from the 4H3 to the transition state (2,5B) binding conformation before reacting and adopting a covalent 5S1 conformation. The fluorescent Cy5 ABP 2 labels rIDUA and allows monitoring the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.