title: GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels.
abstract: Many phosphorylation signal transduction pathways in the eukaryotic cell are modulated by posttranslational modification of specific serines/threonines with N-acetylglucosamine (O-GlcNAc). Levels of O-GlcNAc on key proteins regulate biological processes as diverse as the cell cycle, insulin signaling, and protein degradation. The two enzymes involved in this dynamic and abundant modification are the O-GlcNAc transferase and O-GlcNAcase. Structural data have recently revealed that the O-GlcNAcase possesses an active site with significant structural similarity to that of the human lysosomal hexosaminidases HexA/HexB. PUGNAc, an O-GlcNAcase inhibitor widely used to raise levels of O-GlcNAc in human cell lines, also inhibits these hexosaminidases. Here, we have exploited recent structural information of an O-GlcNAcase-PUGNAc complex to design and synthesize a glucoimidazole-based inhibitor, GlcNAcstatin, which is a 5 pM competitive inhibitor of enzymes of the O-GlcNAcase family, shows 100000-fold selectivity over HexA/B, and binds to the O-GlcNAcase active site by mimicking the transition state as revealed by X-ray crystallography. This compound is able to raise O-GlcNAc levels in human HEK 293 and SH-SY5Y neuroblastoma cell lines and thus provides a novel, potent tool for the study of the role of O-GlcNAc in intracellular signal transduction pathways.
authors.affiliation: Division of Biological Chemistry & Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Helge C
authors.initials: HC
authors.lastname: Dorfmueller
authors.forename: Vladimir S
authors.initials: VS
authors.lastname: Borodkin
authors.forename: Marianne
authors.initials: M
authors.lastname: Schimpl
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Shepherd
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Shpiro
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D000215:Acylation; D002460:Cell Line; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases