Yeast based model of anthrax lethal factor toxicity

基于酵母的炭疽致死因子毒性模型

• 批准号: 6953800
• 负责人: METODI V METODIEV
• 金额: $ 13.5万
• 依托单位: UNIVERSITY OF ESSEX
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6953800
• 关键词: Bacillus anthracis; Saccharomyces cerevisiae; anthrax; anthrax toxin; antitoxins; bacterial toxicology; biological models; biotechnology; bioterrorism /chemical warfare; combinatorial chemistry; complementary DNA; cytotoxicity; drug discovery /isolation; drug screening /evaluation; enzyme activity; fungal genetics; gene expression; genetic library; genetic manipulation; high throughput technology; human genetic material tag; mitogen activated protein kinase; model design /development; peptide library; small molecule

DESCRIPTION (provided by applicant): The lethal factor of Bacillus anthracis is a Zn-endoproteinase that targets the mitogen activated protein kinase kinase (MEK) family of signaling proteins. The toxin recognizes particular members of this family and cleaves a motif that is required for the interaction of MEKs with their downstream effectors--mitogen-activated protein kinases (MAPKs). As a consequence, multiple MAPK pathways are inhibited, and this impairs the viability of affected cells. The goal of this project is to develop an in vivo screening system that allows for the rapid identification of potent and highly specific therapeutics. Rather than screening for compounds that affect particular aspects of lethal factor function in vitro (e.g., its proteolytic activity of the toxin or substrate affinity), we will select for molecules that protect whole cells from lethal factor toxicity using specially engineered strains of the budding yeast Saccharomyces cerevisiae. The yeast strains will be designed to conditionally express anthrax lethal factor and to require the function of a human MEK for survival. When expression of the lethal factor is turned on, the MEK will be inactivated and the cells will die. By selecting for the viability of yeast cells, we can screen hundreds of thousands of compounds and identify potent anti-toxins in a matter of weeks. An important advantage of this approach is that it requires maintenance of MEK function. Agents that interfere with MEK signaling will not pass through the screen. Another attractive feature of this system is that in comparison to mammalian cell-based screens, yeast allow for a much faster response to new mutant strains of the pathogen due to its relatively rapid rate of growth. In addition to providing proof of concept, we will optimize the yeast-based system for high-throughput screening of peptide and cDNA libraries, as well as combinatorial libraries of membrane-permeable small molecules.

描述（由申请人提供）：炭疽杆菌的致死因子是一种锌内切蛋白酶，其靶向信号蛋白的丝裂原激活蛋白激酶激酶（MEK）家族。该毒素识别该家族的特定成员，并裂解 MEK 与其下游效应器——丝裂原激活蛋白激酶 (MAPK) 相互作用所需的基序。结果，多个 MAPK 途径受到抑制，从而损害受影响细胞的活力。该项目的目标是开发一种体内筛选系统，能够快速识别有效且高度特异性的治疗方法。我们不会筛选影响体外致死因子功能特定方面的化合物（例如毒素的蛋白水解活性或底物亲和力），而是使用专门设计的芽殖酵母菌株来选择保护整个细胞免受致死因子毒性的分子酿酒酵母。酵母菌株将被设计为有条件地表达炭疽致死因子，并需要人类 MEK 的功能才能生存。当致死因子的表达开启时，MEK 将失活，细胞将死亡。通过选择酵母细胞的活力，我们可以在几周内筛选数十万种化合物并鉴定出有效的抗毒素。这种方法的一个重要优点是它需要维持 MEK 功能。干扰 MEK 信号传导的药剂将不会通过屏幕。该系统的另一个吸引人的特点是，与基于哺乳动物细胞的筛选相比，酵母由于其相对较快的生长速度，可以对病原体的新突变株做出更快的反应。除了提供概念验证外，我们还将优化基于酵母的系统，用于肽和 cDNA 文库以及膜渗透性小分子组合文库的高通量筛选。