Gross Benjamin J, Kraybill Brian C, Walker Suzanne
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Am Chem Soc. 2005 Oct 26;127(42):14588-9. doi: 10.1021/ja0555217.
O-GlcNAcylation of serine and threonine residues is a dynamic and essential post-translational modification involved in signaling pathways in eukaryotes. Studies of O-GlcNAcylation would be aided by small-molecule inhibitors of O-GlcNAc transferase (OGT), the sole enzyme know to mediate this modification, but discovery of such molecules has been hampered by poor expression of cloned OGT and lack of suitable high-throughput screens. This Communication describes the development an expression system to access large amounts of the catalytic domain of OGT and the implementation of a fluorescence-based substrate analogue displacement assay that has led to the discovery of a set of OGT inhibitors. This work lays the foundation for both structural and functional analysis of the catalytic domain of OGT.
丝氨酸和苏氨酸残基的O-连接N-乙酰葡糖胺化是一种动态且重要的翻译后修饰，参与真核生物的信号通路。O-连接N-乙酰葡糖胺转移酶（OGT）的小分子抑制剂有助于对O-连接N-乙酰葡糖胺化进行研究，OGT是已知介导这种修饰的唯一酶，但克隆OGT的低表达以及缺乏合适的高通量筛选方法阻碍了此类分子的发现。本通讯描述了一种表达系统的开发，该系统可获得大量OGT催化结构域，并实施了基于荧光的底物类似物置换测定法，从而发现了一组OGT抑制剂。这项工作为OGT催化结构域的结构和功能分析奠定了基础。
