Identification of the Optimal Structure Required for a Shiga Toxin Neutralizer with Oriented Carbohydrates to Function in the Circulation

Abstract

Shiga toxin (Stx) is a major virulence factor of Stx-producing Escherichia coli. Recently, we developed a therapeutic Stx neutralizer with 6 trisaccharides of globotriaosyl ceramide, a receptor for Stx, in its dendrimer structure (referred to as "SUPER TWIG [1]6") to function in the circulation. Here, we determined the optimal structure of SUPER TWIG for it to function in the circulation and identified a SUPER TWIG with 18 trisaccharides, SUPER TWIG (2)18, as another potent Stx neutralizer. SUPER TWIGs (1)6 and (2)18 shared a structural similarity, a dumbbell shape in which 2 clusters of trisaccharides were connected via a linkage with a hydrophobic chain. The dumbbell shape was found to be required for formation of a complex with Stx that enables efficient uptake and degradation of Stx by macrophages and, consequently, for potent Stx-neutralizing activity in the circulation. We also determined the binding site of the SUPER TWIGs on Stx.

Innovative Solutions to Sticky Situations: Antiadhesive Strategies for Treating Bacterial Infections

“…To date, many successful examples of antivirulence therapeutic studies have focused on abrogating the effectiveness of toxins, be it through direct inhibition of activity, delivery, or attachment to the host cell (18)(19)(20). One such study utilized a glycomimetic approach to inhibit Shiga toxin's recognition of the host receptor, globotriaosylceramide, which has proven to be effective in vitro and in animal models of enterohemorrhagic Escherichia coli infections (21,22). Similarly, the monoclonal antibody raxibacumab targets the protective antigen component of anthrax toxin (23) and was approved by the FDA in 2012 for protection against and treatment of inhaled anthrax (23)(24)(25).…”

Receptor-mimic probiotics: potential therapeutics for bacterial toxin-mediated enteric diseases

“…Earlier studies had shown that simple oligosaccharides mimicking the receptor structure were not particularly potent toxin inhibitors. However, vastly improved toxin binding kinetics were achieved when analogous oligo saccharides were displayed on complex molecular scaffolds that optimized multivalent docking with the B pentamer [2][3][4][5]. At least in some cases, protection in animal [4,5].…”

“…However, vastly improved toxin binding kinetics were achieved when analogous oligo saccharides were displayed on complex molecular scaffolds that optimized multivalent docking with the B pentamer [2][3][4][5]. At least in some cases, protection in animal [4,5]. However, notwithstanding the elegance of the underlying chemistry, such multivalent synthetic ligands are likely to be expensive to produce on a large scale.…”

Structural Analysis of the Interaction between Shiga Toxin B Subunits and Linear Polymers Bearing Clustered Globotriose Residues

“…Finally, we determined the binding sites of various Gb3 polymers on the Stx1 and Stx2 B subunits by using a series of 1BH and 2BH with mutations at the trisaccharide-binding sites (15). Kinetic analysis was performed by using the BIAcore system.…”