Assay development and screening for inhibitors targeting Shiga toxin 2

针对志贺毒素 2 的抑制剂的检测开发和筛选

• 批准号: 10322373
• 负责人: Xiao-Ping Li
• 金额: $ 51.17万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-25 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322373
• 关键词: Active Sites; Acute Kidney Failure; Adenine; Affinity; Antibiotic Therapy; Antibiotics; Antidotes; Apoptosis; Bacteriophages; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Catalytic Domain; Categories; Category B pathogen; Cells; Chemicals; Child; Complex; Cryoelectron Microscopy; Crystallization; Cytolysis; Cytoprotection; Depurination; Development; Diarrhea; Disease; Docking; Drug Targeting; Escherichia coli; Escherichia coli EHEC; Escherichia coli Infections; FDA approved; Glycine decarboxylase; Goals; Hemolytic-Uremic Syndrome; Hemorrhagic colitis; Infant; Infection; Kidney Failure; Kinetics; Libraries; Life; Measures; Molecular; Molecular Structure; Morbidity - disease rate; National Security; Outcome; Pathogenesis; Peptide Fragments; Peptides; Phage Display; Pharmaceutical Chemistry; Protein Synthesis Inhibition; Public Health; RNA, Ribosomal, 28S; Research; Ribosomal Interaction; Ribosomes; Ricin; Risk; Roentgen Rays; Shiga Toxin; Shiga-Like Toxin I; Shiga-Like Toxin II; Shigella; Shigella Infections; Shigella dysenteriae; Site; Solubility; Structure; Therapeutic; Toxic effect; Toxin; Triage; Vaccines; Variant; Virulence Factors; Work; World Health Organization; X-Ray Crystallography; analog; assay development; base; biological adaptation to stress; cytotoxicity; design; drug discovery; foodborne pathogen; inhibitor; insight; lipophilicity; mortality; mutant; nanomolar; novel; novel strategies; pathogen; physical property; prevent; public health priorities; screening; small molecule inhibitor; structural biology; therapeutic development; therapeutically effective; tool

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.

摘要 产志贺毒素(STX)的大肠杆菌(STEC)和志贺氏菌痢疾杆菌是食源性致病菌 这可能会导致严重的发病率和死亡率。STEC感染可进展为 出血性结肠炎(HC)或危及生命的溶血性尿毒症综合征(HUS)。HUS是最多的 美国儿童急性肾功能衰竭的常见原因。目前还没有FDA批准的 针对STEC或志贺氏菌感染的疫苗或治疗药物。此外，抗生素的使用 会加重疾病。STEC和志贺氏菌被列为国家B类病原菌 安全和公共卫生风险。志贺毒素一直是一个独特的具有挑战性的药物靶点。小的 志贺毒素具有高效酶活性的分子抑制剂尚未被鉴定。 A1亚基与核糖体的相互作用以前没有被认为是一种潜在的药物 目标。这项提议的目标是通过开发新的屏幕来填补这一空白，以识别 片断和多肽抑制剂，通过抑制Stx2与细胞外信号通路的相互作用来阻断其活性 核糖体。我们确定P茎是STX1(Stx1A1)A1亚基的核糖体对接位点。 和Stx2(Stx2A1)，并显示与保守的最后11位相对应的11位肽 P蛋白残基与Stx2A1结合并抑制其活性。这些研究证实了毒素- 核糖体相互作用作为抑制剂发现的新靶点。我们进行了一次初步的 片段筛选并鉴定出与Stx2A1具有微摩尔亲和力的片段。在目标1中 我们建议开发基于Biacore的初级筛查来识别片段，这些片段与 Stx2A1具有较高的亲和力。我们将使用活动分析验证命中，并验证绑定和 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 将用于将所选片段优化为更有效的线索 Stx2的实验X射线晶体结构。在目标2中，我们将开发噬菌体展示 多个拷贝的P蛋白多肽，以确定该多肽基序是否多价展示 会破坏Stx2A1与核糖体的相互作用。我们将随机筛选P7噬菌体 展示文库以确定比天然P更能与Stx2A1结合的新多肽 蛋白多肽并抑制其活性。在目标3中，我们提出了解决低温电磁结构的建议 Stx2与核糖体形成复合体，便于鉴定P蛋白的结合部位 缓蚀剂的优化。我们希望我们独特的分析方法能解剖毒素核糖体 结合药物化学和结构生物学的相互作用和毒素活性 专业知识将导致开发新的工具化合物和多肽，这可以提供 对STEC发病机制的生化和机制洞察。