The Design of Inhibitors of Anthrax Toxin

描述(申请人提供)：炭疽病是由炭疽芽孢杆菌引起的。在气雾剂中传播孢子的能力和吸入性炭疽病的高死亡率导致了将孢子用作生物武器。吸入性炭疽的抗生素治疗在感染后期可能无效，因为血液中高水平的毒素会导致死亡。我们的研究计划集中在炭疽毒素抑制剂的开发上，炭疽毒素是细菌分泌的三种蛋白质的组合。保护性抗原(PA)与受体结合，并被蛋白酶切割，允许细胞相关的PA63片段的七聚异构化。肝细胞异构化使酶毒素成分、水肿因子(EF)和致死因子(LF)结合，并触发这些复合体的内吞作用。内体的酸性环境导致酶蛋白转移到胞浆中，在那里它们发挥其毒性作用。EF是一种腺苷环化酶，通过多种机制损害先天免疫反应。LF是一种导致巨噬细胞裂解的蛋白酶，巨噬细胞裂解会导致休克样症状和死亡。最近发现的炭疽毒素受体(ATR)将有助于开发抑制炭疽毒素作用的分子。我们将使用两种方法来分离PA-ATR相互作用的抑制剂，并在体外测试这些抑制剂阻止中毒过程的能力。在第一种方法中，我们将选择与ATR结合的多肽，然后将该多肽的多个拷贝连接到聚合物骨架上。预测得到的多价化合物与ATR结合的亲和力比仅有多肽更高，并防止PA与细胞结合。第二种方法是基于我们之前观察到的ATR的可溶性片段在体外可以保护细胞免受毒素的影响。我们假设，与PA结合的突变片段比野生型亲和力更高，将是更有效的抑制剂。我们将通过连续几轮的随机诱变、选择和重组来分离这个突变体。这些化合物可能会延长炭疽病成功治疗的时间。

DESCRIPTION (provided by applicant): Anthrax is caused by the spore-forming bacterium Bacillus anthracis. The ability to deliver the spores in an aerosol and the high mortality rate of inhalational anthrax have led to the use of the spores as a biological weapon. Antibiotic treatment of inhalational anthrax can be ineffective late in the infection because high levels of toxin in the blood cause death. Our research proposal is focused on the development of inhibitors of anthrax toxin, a combination of three proteins secreted by the bacterium. Protective antigen (PA) binds a receptor and is cleaved by a protease, allowing the heptamerization of the cell-associated PA63 fragment. Heptamerization allows binding of the enzymatic toxin components, edema factor (EF) and lethal factor (LF), and triggers endocytosis of these complexes. The acidic environment of the endosome leads to the translocation of the enzymatic proteins to the cytosol where they exert their toxic effects. EF is an adenylate cyclase that impairs the innate immune response by a variety of mechanisms. LF is a protease that causes lysis of macrophages, which results in shock-like symptoms and death. The recent identification of the anthrax toxin receptor (ATR) will facilitate the development of molecules that inhibit anthrax toxin action. We will use two approaches to isolate inhibitors of the PA-ATR interaction and test these inhibitors for the ability to block the intoxication process in vitro. In the first approach, we will select a peptide that binds ATR and then attach multiple copies of this peptide to a polymeric backbone. The resulting polyvalent compound is predicted to bind ATR with higher affinity than the peptide alone and prevent binding of PA to cells. The second approach is based on our previous observation that a soluble fragment of ATR can protect cells from toxin in vitro. We hypothesize that a mutant fragment that binds PA with higher than wild-type affinity will be a more effective inhibitor. We will isolate this mutant through sequential rounds of random mutagenesis, selection, and recombination. These compounds may extend the time during which a case of anthrax can be treated successfully.