Basic Research for development of new drug against Shiga-toxin producing E.Coli infection

滋贺毒素（Stx）是产Stx大肠杆菌的主要毒力因子。最近，我们开发了一种治疗性Stx中和剂，其树枝状结构中具有6个三羟甲基的球状神经酰胺（Gb3），Stx的受体（称为“SUPER TWIG [1]6”），以在循环中发挥作用。在这里，我们确定了SUPER TWIG的最佳结构，使其在循环中发挥作用，并确定了一种具有18个三联体的SUPER TWIG，SUPER TWIG（2）18，作为另一种有效的Stx中和剂。SUPER TWIGs（1）6和（2）18具有结构相似性，即哑铃形状，其中2个三聚体簇通过与疏水链的键连接。发现哑铃形状是与Stx形成复合物所需的，该复合物能够使Stx被巨噬细胞有效摄取和降解，因此，在循环中具有有效的Stx中和活性。我们以前还开发了线性聚合物轴承成簇三聚体的Gb3作为口服适用的Stx中和剂。在这里，使用具有将三糖束缚到核心的短间隔区的Gb3聚合物，我们发现缩短间隔区长度显著降低了对Stx 2的结合亲和力，但对Stx 1没有。此外，突变分析显示，末端三核苷酸的必需结合位点在Stx1和Stx2之间完全不同。因此，我们提供了关于治疗性Stx中和剂的最佳结构的信息。

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 (Gb3), 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 previously developed linear polymers bearing clustered trisaccharides of Gb3 as orally applicable Stx neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. Thus, we provided the information about the best structure for the therapeutic Stx neutralizer.