In vivo inactivation of glycosidases by conduritol B epoxide and cyclophellitol as revealed by activity‐based protein profiling

Autor:	Marta Artola, Herman S. Overkleeft, Annemarie H. Meijer, Anthony H. Futerman, Lindsey T. Lelieveld, Ayelet Vardi, Iris Zoutendijk, Mina Mirzaian, Wouter W. Kallemeijn, Chi-Lin Kuo, Johannes M. F. G. Aerts, Herman P. Spaink
Jazyk:	angličtina
Rok vydání:	2019
Předmět:	0301 basic medicine Cell Gaucher disease Biochemistry activity‐based probes 03 medical and health sciences Mice 0302 clinical medicine In vivo medicine Animals Humans Glycoside hydrolase Glycoside Hydrolase Inhibitors Molecular Biology Zebrafish Enzyme Assays Chemistry glucocerebrosidase beta-Glucosidase Activity-based proteomics Brain Parkinson Disease Cell Biology Original Articles Cyclohexanols Glucosylceramidase Isoenzymes Disease Models Animal Kinetics 030104 developmental biology Cyclophellitol medicine.anatomical_structure HEK293 Cells cyclophellitol 030220 oncology & carcinogenesis Larva Original Article Conduritol B-epoxide Lysosomes Glucocerebrosidase conduritol B epoxide Inositol
Zdroj:	The Febs Journal FEBS Journal FEBS Journal, 286(3), 584-600
ISSN:	1742-4658 1742-464X
Popis:	Glucocerebrosidase (GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity‐based probes to visualize catalytic pocket occupancy of candidate off‐target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α‐glucosidase were identified as major off‐targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD‐like models. Enzymes Glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html), nonlysosomal β‐glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html); cytosolic β‐glucosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/21.html); α‐glucosidases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/20.html); β‐glucuronidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/31.html). In vivo target engagement of mechanism‐based glucocerebrosidase (GBA) inhibitors—conduritol B epoxide (CBE) and cyclophellitol (CP)—were examined in cultured cells, zebrafish larvae and mice by competitive activity‐based protein profiling (ABPP). This method utilizes suicide fluorescent enzyme reporter molecules to assess active site occupancy of target glycosidases by inhibitors. The in vivo targets of CBE and CP and their selectivity towards GBA were revealed.
Databáze:	OpenAIRE
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