SOLUBLE RECEPTOR ANALOGS TO INHIBIT VEROTOXIN BINDING

描述 此应用程序用于设计高亲和力的可溶类似物 VT受体糖脂，球三糖神经酰胺(Gb3)，它可以 与儿童肾脏靶细胞表面Gb3有效竞争 从而防止全身VT绑定以启动HUS跟随 胃肠道感染的VT产生性大肠杆菌(VTEC)。我们 已经使用了基于x射线晶体的分子建模的组合 B亚基五聚体的结构，不同的VT与 脱氧-Gb3类似物及其突变位点分析 受体结合，以确定VerooxB内的Gb3结合部位 亚单位。该位点位于相邻亚基之间的缝隙中 包括Aspl7、Glu28、Thr21和Glu65。使用这个模型，我们有 确定了结合糖与氨基之间的氢键网络 受体部位的酸性物质。三种氨基酸的羧基 尤其牵涉其中。半乳糖类似物的合成 在这些化合物涉及的羟基上含有氨基取代 相互作用应该产生具有更高VT结合亲和力的Gb3类似物。 虽然VT/Gb3结合是由Galabiose部分介导的，但游离的 寡糖是一种极差的VT/Gb3结合抑制剂，表明 脂部分在结合中的重要作用。我们的研究表明 这是由于对相对脂肪/碳水化合物的影响 构象。我们设计了一种新的合成策略来使 糖脂模拟其中糖脂的脂类部分 用体积大的刚性疏水取代基截断和衍生。 与游离低聚糖不同，这种可溶的Gb3模拟物是有效的 VT/Gb3结合的体外抑制剂。我们建议优化耦合 VT与这些可溶性受体相互作用的程序和特征 模仿。此外，氨基衍生受体的组合 糖脂模拟形式的碳水化合物类似物很可能 提供极强的VT/膜Gb3结合抑制剂。这个 这一应用的具体目的是开发有效的可溶性 VT体外对膜Gb3结合的拮抗作用 VT介导的敏感培养细胞的细胞毒作用及犬模型 是HUS的。我们的长期目标是建立一种治疗方式 给予这种可溶性的、基于Gb3的竞争性受体类似物， 诊断为婴儿VTEC所致急性腹泻。及时 给予此类类似物应防止肾靶向任何 全身性室性心动过速，否则可能引发内皮损伤，从而 导致肾梗塞和HUS的严重后遗症。

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.