SOLUBLE RECEPTOR ANALOGS TO INHIBIT VEROTOXIN BINDING

抑制维罗毒素结合的可溶性受体类似物

• 批准号: 2017641
• 负责人: CLIFFORD A LINGWOOD
• 金额: $ 9.17万
• 依托单位: HOSPITAL FOR SICK CHLDRN (TORONTO)
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-05 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2017641
• 关键词: Escherichia coli; analog; antitoxins; bacterial toxins; ceramides; chemical binding; chemical substitution; chemical synthesis; cytotoxicity; diarrhea; enzyme linked immunosorbent assay; glycolipids; high performance liquid chromatography; laboratory mouse; laboratory rabbit; nuclear magnetic resonance spectroscopy; oligosaccharides; receptor binding

DESCRIPTION This application is for the design of high affinity soluble analogs of the verotoxin (VT) receptor glycolipid, globotriaosylceramide(Gb3) which can efficiently compete with surface Gb3 of pediatric renal target cells and thereby prevent systemic VT from binding to initiate HUS following gastrointestinal infection with VT producing Escherichia coli (VTEC). We have used a combination of molecular modelling, based on the x-ray crystal structure of the B subunit pentamer, binding of the different VTs to deoxy-Gb3 analogs and analysis of site specific mutations which alter receptor binding, to define the Gb3 binding site within the verotoxin B subunit. This site lies in the cleft between adjacent subunits and comprises Aspl7, Glu28, Thr21, and Glu65. Using this model we have determined the hydrogen bonding network between the bound sugar and amino acids within the receptor site. The carboxyl groups of three amino acids are particularly heavily involved. The synthesis of galabiose analogs containing amino substitutions at the hydroxyl groups involved in these interactions should generate analogs of Gb3 with higher VT binding affinity. Although the VT/Gb3 binding is mediated by the galabiose moiety, the free oligosaccharide is an extremely poor inhibitor of VT/Gb3 binding, indicating the important role of the lipid moiety in binding. Our studies indicate that this is due to an effect on the relative lipid/carbohydrate conformation. We have designed a new synthetic strategy to make soluble glycolipid mimics in which the lipid moiety of the glycolipid has been truncated and derivatized with bulky, rigid hydrophobic substituents. Unlike the free oligosaccharide, such soluble Gb3 mimics are potent inhibitors of VT/Gb3 binding in vitro. We propose to optimize the coupling procedure and characterize the interaction of VT with these soluble receptor mimics. In addition, the combination of amino derivatized receptor carbohydrate analogs in a soluble glycolipid-mimetic format will likely provide extremely potent inhibitors of VT/membrane Gb3 binding. The specific aims of this application are the development of potent soluble inhibitors capable of prevention of membrane Gb3 binding by VT in vitro assay, VT mediated cytotoxicity in sensitive cultured cells and a dog model of HUS. Our long-term goals are to establish a therapeutic modality for the administration of such soluble, Gb3-based, competitive receptor analogs, following diagnosis of acute VTEC-induced diarrhea in infants. Timely administration of such analogs should prevent the renal targeting of any systemic VT which might otherwise initiate the endothelial damage which can result in renal infarct and the severe sequelae of HUS.

描述 本申请用于设计本发明的高亲和力可溶性类似物， verotoxin（VT）受体糖脂，globotriaosylceramide（Gb 3），其可 与小儿肾靶细胞的表面Gb 3有效竞争， 从而防止系统性VT结合引发HUS， 产VT大肠杆菌（VTEC）的胃肠道感染。 我们 使用了基于X射线晶体的分子模型 B亚基五聚体的结构，不同VT与 脱氧-Gb 3类似物和分析位点特异性突变 受体结合，以定义维罗毒素B内的Gb 3结合位点 亚单位 该位点位于相邻亚基之间的裂缝中， 包含Asp 17、Glu 28、Thr 21和Glu 65。 使用这个模型，我们有 确定了结合糖和氨基之间的氢键网络 受体位点内的酸。 三种氨基酸的羧基 参与的特别多。 半乳糖类似物的合成 在涉及这些的羟基上含有氨基取代基， 相互作用应产生具有更高VT结合亲和力的Gb 3类似物。 尽管VT/Gb 3结合是由半乳糖部分介导的，但游离的半乳糖部分是由半乳糖部分介导的。 寡糖是VT/Gb 3结合的极弱抑制剂，表明 脂质部分在结合中的重要作用。 我们的研究表明 这是由于对相对脂质/碳水化合物的影响， 构象 我们设计了一种新的合成策略， 糖脂模拟物，其中糖脂的脂质部分已经 用大体积的刚性疏水取代基截短和衍生化。 与游离寡糖不同，这种可溶性Gb 3模拟物是有效的 VT/Gb 3体外结合抑制剂。 我们建议优化耦合 程序和表征VT与这些可溶性受体的相互作用 模仿者 此外，氨基衍生化受体的组合 可溶性糖脂模拟物形式的碳水化合物类似物可能 提供VT/膜Gb 3结合的极其有效的抑制剂。 的 本申请的具体目的是开发有效的可溶性 能够在体外阻止VT与膜Gb 3结合的抑制剂 试验，敏感培养细胞和犬模型中VT介导的细胞毒性 的HUS。 我们的长期目标是建立一种治疗模式， 施用这种可溶的、基于Gb 3的竞争性受体类似物， 诊断为婴儿急性VTEC诱导的腹泻。 及时 施用此类类似物应防止任何类似物的肾靶向。 全身性VT，否则可能引发内皮损伤， 导致肾梗死和严重的HUS后遗症。