Identifying Host-Factors Required for Toxin Entry and Toxicity

确定毒素进入和毒性所需的宿主因子

• 批准号: 7928785
• 负责人: Erik Hett
• 金额: $ 5.22万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7928785
• 关键词: Affinity Chromatography; Anthrax disease; Antibiotic Resistance; Bacillus anthracis; Biochemical; Biological; Biological Assay; Biology; Botulism; Breathing; Case Fatality Rates; Cell Death; Cell Death Process; Cell physiology; Cells; Cessation of life; Chemicals; Cholera; Communicable Diseases; Development; Diphtheria; Disease; Event; Goals; Immune system; Integration Host Factors; Lead; Membrane Microdomains; Methods; Modeling; Modern Medicine; Molecular; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Process; Proteins; RNA Interference; Research; Role; Signal Pathway; Staging; System; Testing; Tetanus; Toxic effect; Toxin; Virulence Factors; Work; anthrax lethal factor; anthrax toxin; base; cellular targeting; chaperonin; chemical genetics; combat; design; high throughput screening; inhibitor/antagonist; insight; macrophage; novel; novel therapeutics; pathogen; public health relevance; receptor mediated endocytosis; small molecule; therapeutic target; tool; trafficking

DESCRIPTION (provided by applicant): Many bacterial pathogens utilize toxins to cause serious disease. For instance, anthrax lethal toxin (LeTx) is one of the primary virulence factors that Bacillus anthracis utilizes to subvert the host immune system and induce cell death, resulting in a rapid onset of death that is often untreatable with modern medicine, as demonstrated by the 45 percent case-fatality rate among patients with inhalation anthrax during the 2001 attacks. There is an urgent need for a better understanding of the mechanism of anthrax LeTx and corresponding drugs to inhibit these processes. I propose to identify host cellular factors required for toxin entry and toxin-induced cell death using a LeTx macrophage viability model and the complementary approaches of chemical genetics and RNAi. I will conduct a chemical high-throughput screen in macrophages to identify small molecule inhibitors of LeTx-induced cell death. I will prioritize inhibitors according to their potency and activities in secondary assays designed to determine what host pathways or events are inhibited, such as those involved in toxin cell entry or toxicity once the toxin enters the cell. Small molecules with known host targets will be further evaluated using RNAi and structurally different compounds that target the same protein. Alternatively, the best compounds with no known target will be applied to affinity chromatography to identify their host targets, which will help determine their mechanisms of action. We have successfully conducted pilot chemical and RNAi HTS assays that have identified promising leads, indicating that larger screens will likely succeed. The long-term goal of this application is to gain insight into the general biology of toxin translocation and toxicity mechanisms, and the specific biology of anthrax toxin. Accomplishing the proposed aims will illuminate the molecular basis of LeTx entry and toxicity in macrophages and yield small molecules that will serve both as tools to probe LeTx biology as well as proof-of-concept leads for the development of novel therapeutics. This research may lead to new paradigms in host-targeted therapeutics that circumvent the growing problem of antibiotic resistance of important infectious diseases. PUBLIC HEALTH RELEVANCE: Many bacterial pathogens produce toxins that cause serious disease including cholera, tetanus, botulism, diphtheria, and anthrax and often share key steps in cell entry and cell death processes that rely on host proteins. I propose to use an anthrax lethal toxin-induced cell death model to identify host proteins and pathways co-opted by toxins. This work will illuminate the molecular mechanisms underlying resultant pathology and may provide clues for how to combat toxin-related diseases.

描述（由申请人提供）：许多细菌病原体利用毒素引起严重疾病。例如，炭疽致死毒素（LeTx）是炭疽杆菌用来破坏宿主免疫系统并诱导细胞死亡的主要毒力因子之一，导致现代医学通常无法治疗的快速死亡，如2001年袭击期间吸入炭疽患者中45%的病死率所证明的那样。目前迫切需要更好地了解炭疽LeTx的机制和相应的药物来抑制这些过程。我建议使用LeTx巨噬细胞活力模型和化学遗传学和RNAi的互补方法来确定毒素进入和毒素诱导的细胞死亡所需的宿主细胞因子。我将在巨噬细胞中进行化学高通量筛选，以确定LeTx诱导细胞死亡的小分子抑制剂。我将根据抑制剂在二级检测中的效力和活性对抑制剂进行优先排序，二级检测旨在确定哪些宿主途径或事件受到抑制，例如参与毒素细胞进入或毒素进入细胞后的毒性。具有已知宿主靶标的小分子将使用RNAi和靶向相同蛋白质的结构不同的化合物进行进一步评估。或者，没有已知靶点的最佳化合物将被应用于亲和色谱以鉴定其宿主靶点，这将有助于确定其作用机制。我们已经成功地进行了试点化学和RNAi HTS测定，已经确定了有希望的线索，这表明更大的屏幕可能会成功。该应用程序的长期目标是深入了解毒素转运和毒性机制的一般生物学以及炭疽毒素的特定生物学。实现所提出的目标将阐明LeTx进入巨噬细胞和毒性的分子基础，并产生小分子，这些小分子将作为探测LeTx生物学的工具以及开发新疗法的概念验证线索。这项研究可能会导致宿主靶向治疗的新范例，从而规避重要传染病日益严重的抗生素耐药性问题。公共卫生相关性：许多细菌病原体产生毒素，导致严重的疾病，包括霍乱，破伤风，肉毒杆菌中毒，白喉和炭疽，并经常分享细胞进入和细胞死亡过程中的关键步骤，依赖于宿主蛋白质。我建议使用炭疽致死毒素诱导的细胞死亡模型，以确定宿主蛋白质和毒素增选的途径。这项工作将阐明导致病理学的分子机制，并可能为如何对抗毒素相关疾病提供线索。