Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome

Abstract

Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell. Due to the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell surface glycans. As an example of the functional consequences, the inhibitors drastically reduce expression of the sialylated and fucosylated ligand Sialyl Lewis X on myeloid cells, resulting in loss of binding to selectins and impaired leukocyte rolling.

Tailoring recombinant protein quality by rational media design

“…The strategy consisted in attaching a fluorine atom proximal to the endocyclic oxygen, as these compounds are readily converted to the corresponding donor substrate analogs within the cell. The 2-fluor-fucose analog substantially reduced core fucosylation of N-linked glycans in CHO cells at concentrations of about 30-500 µM, thus revealing its inhibitory effect on FucT-VIII 112 . Likewise, the non-sugar related compounds, Cibracon Blue 3GA (K i = 11 µM) and Reactive Red 120 (K i = 2 µM) specifically inhibit FucT-VIII 113 .…”

“…However, the high negative charge of nucleotide sugar analogs prevents them from efficiently crossing cell membranes and, with a few exceptions, questions their utility in cell culture 112 . Much simpler in structure, gallic acid and its derivatives efficiently inhibit FucT-VII in the presence of 10-15 mM Mn 2+ 116 .…”

“…As a result, selective inhibition of sialic acid addition by adding a fluorinated sialic acid analog increases fucosylation 112 . Due to its effective inhibition of steps occurring prior to fucose attachment, kifunensine effectively retains oligomannose residues and hence allows to express non fucosylated proteins at concentrations beyond 60 ng/mL 91 .…”

“…2-naphthyl-2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside specifically and strongly inhibited β4-GalT1 at a concentration of 0.5 mM in human and mouse cell homogenates and bovine serum (FBS), while it does not affect the activity of related enzymes 132 . The use of peracetylated fluorosugars, which successfully inhibited both fucosylation and sialylation, is suggested as UDP-galactose analogs to inhibit GalT 112 . The inhibition constant of UDP-2FGal was found to 2.1.…”

“…The axial 3-fluor-N-acetyl-neuramic acid (3F ax -Neu5Ac) extensively inhibits SiaT in vivo. The orientation of the fluor atom is pivotal, as the equatorial analog (3F eq -Neu5Ac) has no inhibitory effect 112 . In a fed-batch CHO culture expressing a Fc-fusion protein, the addition of 1-50 mg/L of hydrocortisone increased the proportion of sialic acid moieties up to a level 2.5 times higher than the control, which corresponds to a 5-fold increase of acidic isoforms 64 .…”