Cell-Penetrant, Nanomolar O-GlcNAcase Inhibitors Selective against Lysosomal Hexosaminidases

Posttranslational modification of metazoan nucleocytoplasmic proteins with N-acetylglucosamine ( O-GlcNAc) is essential, dynamic, and inducible and can compete with protein phosphorylation in signal transduction. Inhibitors of O-GlcNAcase, the enzyme removing O-GlcNAc, are useful tools for studying the role of O-GlcNAc in a range of cellular processes. We report the discovery of nanomolar OGA inhibitors that are up to 900,000-fold selective over the related lysosomal hexosaminidases. When applied at nanomolar concentrations on live cells, these cell-penetrant molecules shift the O-GlcNAc equilibrium toward hyper- O-GlcNAcylation with EC 50 values down to 3 nM and are thus invaluable tools for the study of O-GlcNAc cell biology. ► Structure-guided design of human O-GlcNAcase inhibitors, GlcNAcstatins ► The GlcNAcstatins are competitive, nanomolar inhibitors ► The molecular basis of the exquisite selectivity revealed by crystallography ► First direct evidence of O-GlcNAcase inhibitors penetrating cells