A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase.
enzymes
fluorescent probes
glycosylation
high-throughput screening
inhibitors
Journal
Angewandte Chemie (International ed. in English)
ISSN: 1521-3773
Titre abrégé: Angew Chem Int Ed Engl
Pays: Germany
ID NLM: 0370543
Informations de publication
Date de publication:
08 06 2020
08 06 2020
Historique:
received:
13
01
2020
revised:
13
02
2020
pubmed:
25
2
2020
medline:
25
3
2021
entrez:
25
2
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32092778
doi: 10.1002/anie.202000621
doi:
Substances chimiques
N-Acetylglucosaminyltransferases
EC 2.4.1.-
O-GlcNAc transferase
EC 2.4.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
9601-9609Informations de copyright
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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