Development of orally active inhibitors of protein and cellular fucosylation

Abstract

The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDPfucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy

“…36 In agreement with those results, addition of 100, 200, and 400 mM resulted in a dramatic reduction of core fucose incorporation into IgG, dropping from 76% fucosylation to 14%, 8%, and 6%, respectively (supplemental Figure 4). For the following experiments, the 76% fucosylated sample was used as anti-TNP high fucose, and the 6% fucosylated sample was used as anti-TNP low fucose.…”

“…36 Antibodies were purified on a protein A (WT IgG1) HiTrap HP column (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) using the Acta Prime Plus system (GE Healthcare Life Sciences) and dialyzed against phosphate-buffered saline (PBS) overnight. IgG-Fc glycosylation was determined by mass spectrometry.…”

Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

“…8B). As 6-alkynyl fucose incorporation was more efficiently performed in living cells onto EGF-like repeats and trombospondin repeats (TSRs) than in N-linked glycans (34,46), its detection could give a good overview of Pofut1 enzymatic activity. Furthermore, costaining with GRP94 allowed us to discriminate O-fuco- sylation occurring in the ER from fucosylation taking place in Golgi apparatus (for mucins and N-glycans) (Fig.…”

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…utilized small molecules to inhibit antibody fucosylation 89 . 2-fluorofucose and 5-alkynylfucose were shown to generate afucosylated monoclonal antibodies.…”