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

基本信息

批准号：
9761636
负责人：
Xiao-Ping Li
金额：
$ 52.66万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-25 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9761636
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 Treatment Efficacy 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/antagonist insight lipophilicity mortality mutant nanomolar novel novel strategies pathogen physical property prevent public health priorities screening small molecule inhibitor structural biology therapeutic development tool

项目摘要

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.

抽象的产生志贺毒素 (Stx) 的大肠杆菌 (STEC) 和志贺氏痢疾杆菌是食源性病原体这可能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）或危及生命的溶血尿毒症综合征（HUS）。 HUS是最美国儿童急性肾衰竭的常见原因。目前尚无 FDA 批准针对 STEC 或志贺氏菌感染的疫苗或治疗方法。此外，使用抗生素加剧病情。 STEC和志贺氏菌被列为国家B类病原体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标。小的尚未鉴定出具有高效力的志贺毒素酶活性分子抑制剂。 A1 亚基与核糖体的相互作用之前尚未作为潜在药物进行过研究目标。该提案的目标是通过开发新颖的屏幕来识别来填补这一空白片段和肽抑制剂通过抑制 Stx2 与核糖体。我们确定 P 茎是 Stx1 (Stx1A1) A1 亚基的核糖体对接位点和 Stx2 (Stx2A1)，并表明对应于保守的最后 11 个的 11 聚体肽 P 蛋白的残基与 Stx2A1 结合并抑制其活性。这些研究确定了毒素核糖体相互作用作为抑制剂发现的新目标。我们进行了初步的片段筛选并鉴定出以微摩尔亲和力与 Stx2A1 结合的片段。目标1 我们建议开发基于 Biacore 的初级筛选来识别片段，这些片段结合 Stx2A1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。药物化学将用于根据所选片段将其优化为更有效的先导化合物使用 Stx2 进行实验 X 射线晶体结构。在目标 2 中，我们将开发噬菌体展示 P 蛋白肽的多个拷贝，以确定该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 噬菌体展示库来鉴定与 Stx2A1 结合比天然 P 更强的新型肽蛋白肽并抑制其活性。在目标 3 中，我们建议解决以下问题的冷冻电镜结构： Stx2 与核糖体复合，识别 P 蛋白的结合位点，以促进抑制剂的优化。我们期望我们独特的分析方法能够解剖毒素核糖体与药物化学和结构生物学相结合的相互作用和毒素活性专业知识将导致新型工具化合物和肽的开发，它们可以提供对 STEC 发病机制的生化和机制洞察。