New Irreversible α‐l‐Iduronidase Inhibitors and Activity‐Based Probes

Abstract

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 of 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 structures of IDUA when complexed with the inhibitors in a non‐covalent transition state mimicking form and a covalent enzyme‐bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half‐chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.

An overview of activity-based probes for glycosidases

“…Similarly, Artola et al tracked ABP pre-labeled -L-iduronidase (IDUA) uptake by mucopolysaccharidosis (MPD)I and mucolipidosis II (MD)II fibroblasts. In this case, application of mannose-6-phosphate abrogated uptake of recombinant IDUA, confirming that IDUA uptake was dependant on the mannose-6-phosphate pathway [54].…”

Glucocerebrosidase: Functions in and Beyond the Lysosome

“…Subsequently, cyclophellitol aziridine ABPs with attached reporter groups via alkyl or acyl linkers were designed reacting with multiple retaining glycosidases in the same class [55,59]. Cyclophellitol aziridine ABPs labeling α-galactosidases, α-glucosidases, α-fucosidase, α-iduronidase, β-galactosidases, and β-glucuronidase, as well as cyclophellitol ABPs labelling galactocerebrosidase, were designed [60][61][62][63][64][65]. Applications of ABPs are the quantitative detection and localization of glycosidases in cells and tissues, as well as identification and characterization of glycosidase inhibitors by competitive ABP profiling [66,67].…”

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics

“…Subsequently, by variation of the configuration of cyclophellitolaziridine a series of new probes were developed for other retaining glycosidases. Detection of active α-D-galactosidase (Willems et al, 2014a), β-D-galactosidase (Marques et al, 2016b), α-L-fucosidase (Jiang et al, 2015), α-D-glucosidases (Jiang et al, 2016), β-D-glucuronidase (Wu et al, 2017), α-L-iduronidase (Artola et al, 2018) and α-L-arabinofuranosidases (McGregor et al, 2020) was achieved with these types of ABPs. Through changing the reporter group into a biotin, the probes can be used for streptavidin-mediated enrichment, followed by proteomics identification of labeled proteins using LC-MS/MS (Jiang et al, 2016).…”