Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells

Abstract

Glycosyltransferases (GTs) are ubiquitous enzymes that catalyze the assembly of glycoconjugates found throughout all kingdoms of nature. A longstanding problem is the rational design of probes that can be used to manipulate GT activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analogue that inhibits, with high potency both in vitro and in cells, the human GT responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analogue. Treatment of cells with the precursor decreases O-GlcNAc in a targeted manner with a single digit micromolar EC50. This approach to inhibition of GTs should be applicable to other members of this increasingly interesting superfamily of enzymes and enable their manipulation in a biological setting.

Metabolic Inhibition of Sialyl-Lewis X Biosynthesis by 5-Thiofucose Remodels the Cell Surface and Impairs Selectin-Mediated Cell Adhesion

“…Recently, we found a solution to this problem for the glycosyltransferase known as O-GlcNAc transferase. We found that peracetylated 5-thio-Nacetylglucosamine was taken up by a wide variety of cells and assimilated by the GlcNAc salvage pathway to form the nucleotide thiosugar analog of UDP-GlcNAc, which acted in cells to inhibit O-GlcNAc-transferase (31). This result suggested to us that a metabolic feeding approach might be more widely applicable.…”

“…Only peracetylated 5T-Fuc was tested in cells because previous work has established that acetate-protected monosaccharides are more amenable to cell uptake than the parent sugars (15,31). GDP-5T-Fuc was chemoenzymatically prepared from (deprotected) 5T-Fuc, ATP, and GTP with the enzyme L-fucokinase-GDP-Lfucose pyrophosphorylase (FKP) (35), which was used according to a procedure published previously (36), with some modifications.…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…While the synthesized compounds inhibit OGT in the micromolar range, it should be noted that they are inactive when used on living cells (Dorfmueller et al 2011). Another recent study developed a synthetic carbohydrate precursor, 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-d-glucopyranose, which can be successfully converted by the cell to produce UDP5SGlcNAc in cell culture and in vitro models (Gloster et al 2011). UDP-5SGlcNAc is not an efficient substrate for OGT, but excludes binding of UDP-GlcNAc therefore inhibiting OGT function (Gloster et al 2011).…”

“…Another recent study developed a synthetic carbohydrate precursor, 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-d-glucopyranose, which can be successfully converted by the cell to produce UDP5SGlcNAc in cell culture and in vitro models (Gloster et al 2011). UDP-5SGlcNAc is not an efficient substrate for OGT, but excludes binding of UDP-GlcNAc therefore inhibiting OGT function (Gloster et al 2011). It is important to note that these inhibitors are not yet commercially available.…”

Metabolic Inhibitors of O‐GlcNAc Transferase That Act In Vivo Implicate Decreased O‐GlcNAc Levels in Leptin‐Mediated Nutrient Sensing

“…They also exhibit off‐target cellular toxicity 5b. Another approach5c has been to generate a GlcNAc analogue, 2‐acetamido‐2‐deoxy‐5‐thio‐α‐ d ‐glucopyranose (5SGlcNAc, 3 ), which in its per‐O‐acetylated form (Ac 4 5SGlcNAc, 3 ‐OAc) can diffuse across the plasma membrane. Within cells, 3 ‐OAc is de‐O‐acetylated and assimilated via the GlcNAc salvage pathway (Figure 1) to generate UDP‐5SGlcNAc ( 4 ), which is a competitive OGT inhibitor ( K i =8 μ m ).…”