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. Because of 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 substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.

Figure 1. Fluorinated Monosaccharide Analogs act as Metabolic Glycosyltransferase Inhibitors

Peracetylated Analogs of Fucose (1-3) or Sialic Acid (7-9) are taken up by cells and converted into the corresponding nucleotide sugars (4-6 and 10-12) through salvage pathways. The fluorinated analogs (5-6, 11-12) act as inhibitors of the corresponding fucosyl- or sialyltransferases and, because they are not transferred, accumulate in the cell. This accumulation then shuts down the de novo synthesis of GDP-Fucose (4) or CMP-NeuAc (10) via a feedback loop, to further decrease fucose or sialic acid expression on the cell surface.

Figure 2. Fluorinated Fucose Analogs act as Fucosyltransferase Inhibitors in Cells

HL-60 or CHO cells were treated with various concentrations of the fucose analogs (1-3). After 3 days, cells were harvested and various fucosylated epitopes were detected with anti-glycan antibodies or lectins via flow cytometry. Inhibition of the FUT4 enzyme was assessed in HL-60 cells with an anti-Lewis X antibody (a). Similarly, FUT7 inhibition was assessed in this cell line, but utilizing the anti-Sialyl Lewis X antibody (b). To analyze FUT8 inhibition, CHO cells and the lectin AAL were used (c). In all cases the data were normalized to cells treated with DMSO only as 100% fucosylated epitope expression and unstained cells as 0%. Data shown is representative of three independent experiments carried out in triplicate.

Figure 3. A Fluorinated Sialic Acid Analogs act as a Sialyltransferase Inhibitor in Cells

HL-60 or Ramos cells were treated with various concentrations of the sialic acid analogs (7-9). After 3 days, cells were harvested and various sialylated epitopes were detected with anti-glycan antibodies or lectins via flow cytometry. Inhibition of the ST3Gal I and/or ST3Gal II enzymes was assessed in HL-60 cells by loss of the sialylated epitope NeuAcα2,3Galβ1,3GalNAcαSer/Thr (Sialyl-T Antigen) and detection of the asialo structure Galβ1,3GalNAcαSer/Thr (T Antigen) by the lectin PNA (a). Decreases in Sialyl Lewis X formation were determined with HL-60 cells and an anti-Sialyl Lewis X antibody reflecting inhibition of ST3Gal IV and/or ST3Gal VI in this cell line (b). Inhibition of NeuAcα2,6Gal formation, the product of ST6Gal I and/or ST6Gal II, was assessed with Ramos cells and the lectin SNA (c). In each case the data represents one of three independent experiments carried out in triplicate. For (a) the expression of the T-antigen was normalized to DMSO only as 0% and the maximum inhibitor treatement as 100%. From this, Sialyl T antigen expression was expressed as 100% - (percent T-antigen expression). For (b,c) the data were normalized to cells treated with DMSO only as 100% sialylated epitope expression and unstained cells as 0%.

Figure 4. Mass spectrometry Analysis of N-glycans from Inhibitor treated cells

HL-60 cells were treated for 7 days with DMSO only, 200 μM (2), 200 μM (8), or 200 μM each of (2) and (8). N-linked glycans from each sample were then isolated, permethylated and analyzed by MALDI-TOF MS. The four spectra represent treatment of the cells with (a) DMSO only, (b) the fucosyltransferase inhibitor (2), (c) the sialyltransferase inhibitor (8), or (d) both inhibitors in concert. All molecular ions are [M+Na]⁺. Putative structures are based on composition, tandem MS, and biosynthetic knowledge. Structures that show sugars outside a bracket have not been unequivocally defined.

Figure 5. Selectin Binding and Selectin-Mediated Leukocyte Rolling

(a) Flow cytometry analysis of HL-60 cells treated for 5 days with 200 μM each of (2) or (8) leads to decreases in anti-Sialyl Lewis X antibody as well as recombinant E- and P-selectin binding. This leads to higher rolling velocities (at 3 dynes/cm²) of these cells on selectin coated surfaces (b) and a decreased shear resistance on E- but not P-selectin coated surfaces (c). *** corresponds to p < 0.0001.