Yeast based model of anthrax lethal factor toxicity

描述(申请人提供)：炭疽芽孢杆菌的致死因子是一种以丝裂原活化蛋白激酶(MEK)家族信号蛋白为靶标的锌内切酶。这种毒素识别这个家族的特定成员，并切割MEKS与其下游效应器--丝裂原激活蛋白激酶(MAPK)相互作用所需的一个基序。结果，多个MAPK通路被抑制，这损害了受影响细胞的生存能力。该项目的目标是开发一种体内筛选系统，允许快速识别有效和高度特异的治疗药物。我们不会在体外筛选影响致死因子功能的特定方面的化合物(例如，毒素的蛋白分解活性或底物亲和力)，而是使用特殊工程的酿酒酵母芽生菌株来筛选保护整个细胞免受致死因子毒性的分子。酵母菌株将被设计成有条件地表达炭疽致命因子，并需要人类MEK的功能才能生存。当致命因子的表达开启时，MEK将被灭活，细胞将死亡。通过选择酵母细胞的活性，我们可以在几周内筛选出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信号的代理将不会通过屏幕。该系统的另一个吸引人的特点是，与哺乳动物细胞筛查相比，酵母对新的病原体突变株的反应要快得多，因为它的生长速度相对较快。除了提供概念验证，我们还将优化基于酵母的系统，用于高通量筛选多肽和cDNA文库，以及膜渗透小分子的组合文库。

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.