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

Abstract

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.

Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity

“…The Vocadlo group published a new bioassay for screening OGT inhibitors in HTS based on a chemically modified UDP-GlcNAc [39]. The modified GlcNAc was prepared by extending the amino group of the corresponding glucosamine precursor with a Bodipy fluorescent group (Figure 11).…”

“…The fluorescent signal is measured to allow direct quantification of glycosylation [40] ( Figure 12). This assay was used to perform a high-throughput screening on a library of known bioactive compounds [39]. The screening led to the discovery of Dingo-4a as a potent OGT inhibitor with IC50 of 6 μM (Figure 13), which was originally developed as a target for dynamic GTPase (Dynl).…”

“…The screening led to the discovery of Dingo-4a as a potent OGT inhibitor with IC50 of 6 μM (Figure 13), which was originally developed as a target for dynamic GTPase (Dynl). This assay was used to perform a high-throughput screening on a library of known bioactive compounds [39]. The screening led to the discovery of Dingo-4a as a potent OGT inhibitor with IC 50 of 6 µM (Figure 13), which was originally developed as a target for dynamic GTPase (Dynl).…”

Bovine Serum Albumin as A Superior “FP-Tag” for High-Throughput Glycosyltransferase Assay

“…23 The Vocadlo group recently reported a novel, high-throughput, direct fluorescent activity assay for OGT based on streptavidin-coated microplates or streptavidin-coated magnetic beads. 24 However, the assay requires washing steps and those microplates or magnetic beads are expensive for large scale HTS campaigns.…”

Bovine Serum Albumin as A Superior “FP-Tag” for High-Throughput Glycosyltransferase Assay

“…23 The Vocadlo group recently reported a novel, high-throughput, direct fluorescent activity assay for OGT based on streptavidin-coated microplates or streptavidin-coated magnetic beads. 24 However, the assay requires washing steps and those microplates or magnetic beads are expensive for large scale HTS campaigns.…”