A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

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...

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Bibliographic Details
Published in:Angewandte Chemie 2020-06, Vol.132 (24), p.9688-9696
Main Authors: Alteen, Matthew G., Gros, Christina, Meek, Richard W., Cardoso, David A., Busmann, Jil A., Sangouard, Gontran, Deen, Matthew C., Tan, Hong‐Yee, Shen, David L., Russell, Cecilia C., Davies, Gideon J., Robinson, Phillip J., McCluskey, Adam, Vocadlo, David J.
Format: Article
Language:English
Subjects:
Assaying
Chemistry
Enzymes
Fluorescence
fluorescent probes
glycosylation
high-throughput screening
Inhibitors
Optimization
Screening
Substrate inhibition
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Summary: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. A robust fluorescence‐based assay accurately reports on the sugar‐transfer activity of glycosyltransferases. This assay is amenable to high‐throughput screening and should be applicable to a diverse set of group‐transfer enzymes. This assay is validated by screening of a library of known bioactive molecules to identify a new O‐GlcNAc transferase antagonist.
ISSN:0044-8249
1521-3757
DOI:10.1002/ange.202000621