Efficient inhibition of O-glycan biosynthesis using the hexosamine analog Ac₅GalNTGc

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that Ac₅GalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, Ac₅GalNTGc increased cell-surface VVA binding by ∼10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry, and western blot analysis of HL-60 promyelocytes demonstrated that 50–80 μM Ac₅GalNTGc prevented elaboration of 30%–60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac₅GalNTGc resulted in 50%–80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. Ac₅GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, Ac₅GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.