A Strategy to Discover Inhibitors of O-Linked Glycosylation

Abstract

Protein O-glycosylation is involved in numerous biological processes. Deconvoluting the functions of O-linked glycans requires having glycosyltransferase (Gtf) inhibitors suitable for use in cells. Although high-throughput screening has provided small-molecule inhibitors suitable for dissecting kinase pathways, it has not been applied to discover inhibitors of protein glycosylation due to a lack of suitable assays. Here we describe a general protease-protection assay in which a peptide labeled with a FRET pair is subjected to glycosylation and then treated with a protease that discriminates between glycosylated and non-glycosylated peptides. We show that this approach can be adapted to monitor the activity of O-GlcNAc transferase (OGT), a Gtf involved in intracellular signaling, as well as two O-GalNAc transferases (ppGalNAc T1 and T2), which initiate O-linked mucin-type glycosylation. We have used the strategy to screen 124 226 small molecules against OGT and have discovered several low micromolar inhibi...