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.

抽象的志贺毒素（STX）产生大肠杆菌（STEC）和Shigella dysenteriae是食源性病原体这可能导致严重的发病率和死亡率。 STEC感染可以发展为出血性结肠炎（HC）或威胁生命的溶血性尿毒症综合征（HUS）。 HUS是最大的美国儿童急性肾衰竭的常见原因。目前尚未批准FDA 针对STEC或Shigella感染的疫苗或治疗剂。而且，使用抗生素加剧了这种疾病。 STEC和Shigella被归类为国家的B类病原体安全和公共卫生风险。 Shiga Toxin一直是一个挑战性的药物靶标。小的尚未鉴定出具有高效力的志贺毒素酶活性的分子抑制剂。 A1亚基与核糖体的相互作用尚未被视为潜在药物目标。该提案的目的是通过开发新颖的屏幕来填补这一空白以识别片段和肽抑制剂通过抑制其与STX2的活性来抑制其与核糖体。我们确定p茎是STX1 A1亚基的核糖体对接位点（STX1A1）和STX2（STX2A1），并表明与保守的最后11个相对应的11-Mer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是抑制剂发现的新目标。我们进行了初步片段屏幕和与sTx2A1具有微摩尔亲和力结合的片段。在目标1中我们建议开发基于BIACOR的主要筛选，以识别与结合的片段 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选择性。药物化学将使用基于它们具有STX2的实验X射线晶体结构。在AIM 2中，我们将开发噬菌体显示 P蛋白肽的多个副本，以确定该肽基序的多价显示会破坏STX2A1与核糖体的相互作用。我们将筛选随机P7噬菌体显示库以比本地P更强烈地识别可以与STX2A1结合的新型肽蛋白质肽并抑制其活性。在AIM 3中，我们建议解决的冷冻EM结构与核糖体配合物中的STX2，以识别P蛋白的结合位点以促进抑制剂的优化。我们希望我们的独特测定法会剖析毒素 - 核糖体相互作用和毒素活性与药物化学和结构生物学结合专业知识将导致新型工具化合物和肽的开发，这些化合物和肽可以提供对STEC发病机理的生化和机械洞察力。