Assay development and screening for inhibitors targeting Shiga toxin 2

摘要 产滋贺毒素（Stx）的E.大肠杆菌（STEC）和志贺氏菌是食源性致病菌 会导致严重的发病率和死亡率。STEC感染可以进展为 出血性结肠炎（HC）或危及生命的溶血性尿毒症综合征（HUS）。HUS是最 急性肾衰竭的常见原因目前，FDA尚未批准 针对STEC或志贺氏菌感染的疫苗或治疗剂。此外，抗生素的使用 会加重病情STEC和志贺氏菌属被列为国家卫生部的B类病原体。 安全和公共卫生风险。滋贺毒素一直是一个独特的具有挑战性的药物靶标。小 尚未鉴定出高效的滋贺毒素酶活性的分子抑制剂。 A1亚基与核糖体的相互作用以前没有被作为潜在的药物进行过研究 目标这项提案的目标是通过开发新的屏幕来填补这一空白， 片段和肽抑制剂，通过抑制Stx 2与 核糖体我们确定P柄为Stx 1的A1亚基（Stx 1A 1）的核糖体对接位点。 和Stx 2（Stx 2A 1），并显示对应于保守的最后11个 P蛋白的残基与Stx 2A 1结合并抑制其活性。这些研究建立了毒素- 核糖体相互作用作为抑制剂发现的新靶点。我们进行了初步的 片段筛选并鉴定以微摩尔亲和力结合Stx 2A 1的片段。在aim 1中 我们建议开发基于Biacore的初步筛选，以识别与 Stx 2A 1具有更高的亲和力。我们将使用活性测定来验证命中，并验证结合， 使用核糖体结合和活性位点突变体的抑制剂的选择性。药物化学 将被用来优化选定的片段，根据其更有效的线索， 实验X射线晶体结构与Stx 2。目标二是发展噬菌体展示技术 P蛋白肽的多个拷贝，以确定该肽基序的多价展示 将破坏Stx 2A 1与核糖体的相互作用。我们将随机筛选P7噬菌体 展示文库以鉴定可以比天然P更强地结合Stx 2A 1的新肽 蛋白肽并抑制其活性。在目标3中，我们提出解决的冷冻EM结构， Stx 2与核糖体复合，以确定P蛋白的结合位点，以促进 抑制剂的优化。我们希望我们独特的分析方法能够解剖毒素-核糖体 结合药物化学和结构生物学， 专业知识将导致开发新的工具化合物和肽，这可以提供 生化和机制的深入了解STEC发病机制。

Abstract Shiga toxin (Stx) producing E. coli (STEC) and Shigella dysenteriae are foodborne pathogens that can cause severe morbidity and mortality. STEC infections can progress to either hemorrhagic colitis (HC) or life-threatening hemolytic-uremic syndrome (HUS). HUS is the most common cause of acute renal failure in US children. Presently there are no FDA approved vaccines or therapeutics against STEC or Shigella infection. Moreover, the use of antibiotics exacerbates the disease. STEC and Shigella are classified as category B pathogens of national security and public health risk. Shiga toxin has been a uniquely challenging drug target. Small molecule inhibitors of Shiga toxin enzymatic activity with high potency have not been identified. Interaction of A1 subunits with ribosomes has not been previously examined as a potential drug target. The goal of this proposal is to fill this gap by developing novel screens to identify fragment and peptide inhibitors that disrupt activity of Stx2 by inhibiting its interaction with the ribosome. We identified P stalk as the ribosome docking site of the A1 subunits of Stx1 (Stx1A1) and Stx2 (Stx2A1) and showed that an 11-mer peptide corresponding to the conserved last 11 residues of P proteins binds to Stx2A1 and inhibits its activity. These studies established toxin- ribosome interactions as a new target for inhibitor discovery. We carried out a preliminary fragment screen and identified fragments that bind to Stx2A1 with micromolar affinity. In aim 1 we propose to develop Biacore-based primary screens to identify fragments, which bind to Stx2A1 with higher affinity. We will validate the hits using activity assays and verify binding and selectivity of the inhibitors using ribosome binding and active site mutants. Medicinal chemistry will be used to optimize the selected fragments into more potent leads based on their experimental X-ray crystal structure with Stx2. In aim 2 we will develop phage displaying multiple copies of the P protein peptide to determine if multivalent display of this peptide motif will disrupt the interaction of Stx2A1 with the ribosome. We will screen random P7 phage display library to identify novel peptides that can bind to Stx2A1 more strongly than the native P protein peptide and inhibit its activity. In aim 3 we propose to solve the cryo-EM structure of Stx2 in complex with the ribosome to identify the binding sites of the P proteins to facilitate optimization of the inhibitors. We expect that our unique assays to dissect toxin-ribosome interactions and toxin activity in combination with the medicinal chemistry and structural biology expertise will lead to the development of novel tool compounds and peptides, which can provide biochemical and mechanistic insight into STEC pathogenesis.