期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 59, 期 24, 页码 9601-9609出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202000621
关键词
enzymes; fluorescent probes; glycosylation; high-throughput screening; inhibitors
资金
- Tier I Canada Research Chair in Chemical Glycobiology
- E.W.R. Steacie Memorial Fellowship
- Canadian Glycomics Network [CD-1]
- Natural Sciences and Engineering Research Council [RGPIN-2015-05426]
- Canadian Foundation for Innovation [33097, 36766, 37241]
- British Columbia Knowledge Development Foundation [662-805223, 862-805636]
- Y.P. Heung Foundation
- PDRA [RP\EA\180016]
- National Health & Medical Research Council Australia [GNT1162515, GNT1137064]
- Australian Research Council [DP180101781]
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.
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