A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase.,Angewandte Chemie International Edition

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A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase.
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2020-02-24 , DOI: 10.1002/anie.202000621
Matthew G Alteen ₁ , Christina Gros ₁ , Richard W Meek ₂ , David A Cardoso ₃ , Jil A Busmann ₄ , Gontran Sangouard ₁ , Matthew C Deen ₁ , Hong-Yee Tan ₁ , David L Shen ₄ , Cecilia C Russell ₅ , Gideon J Davies ₂ , Phillip J Robinson ₃ , Adam McCluskey ₅ , David J Vocadlo _{1,

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Affiliation

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.

中文翻译：

糖基转移酶的直接荧光活性测定法可以方便地进行高通量筛选：应用于O-GlcNAc转移酶。

糖基转移酶在从细菌到人类的物种中执行重要的细胞功能。尽管它们在生物学中起着至关重要的作用，但简单，强大的活性测定方法却很容易开发，这些测定方法可轻松应用于高通量筛选这些酶的抑制剂。本文中，我们报道了一种基于珠的策略，可使用简单的荧光测量方法灵敏地测量糖基转移酶的基团转移活性，而无需偶联酶或二级反应。通过对各种底物和抑制剂进行详细的Michaelis-Menten动力学分析，我们使用O-GlcNAc转移酶（OGT）作为模型系统来验证测定的性能和准确性。通过优化该检测方法并将其应用于高通量筛选，可以筛选OGT抑制剂，导致了新型的抑制性支架。我们相信这种测定方法不仅对OGT的研究具有重要的价值，而且作为筛选糖基转移酶和其他基团转移酶的通用方法也将得到广泛应用。

更新日期：2020-02-24

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