GlcNAcstatin:  a Picomolar, Selective O-GlcNAcase Inhibitor That Modulates Intracellular O-GlcNAcylation Levels

• Published in: Journal of the American Chemical Society 2006-12, Vol.128 (51), p.16484-16485
• Main Authors: Dorfmueller, Helge C, Borodkin, Vladimir S, Schimpl, Marianne, Shepherd, Sharon M, Shpiro, Natalia A, van Aalten, Daan M. F
• Format: Article
• Language: English
• Subjects: Acylation; beta-N-Acetylhexosaminidases - antagonists & inhibitors; beta-N-Acetylhexosaminidases - chemistry; Cell Line; Chemistry; Crystallography, X-Ray; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Exact sciences and technology; General and physical chemistry; Humans; Models, Molecular; Molecular Conformation; Molecular Weight; Structure-Activity Relationship
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja066743n

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.