The Design of Inhibitors of Anthrax Toxin

描述（由申请人提供）：炭疽是由孢子形成细菌炭疽杆菌引起的。 由于炭疽孢子能够以气溶胶的形式传播，而且由于吸入性炭疽的死亡率很高，因此人们将其用作生物武器。 吸入性炭疽的抗生素治疗在感染后期可能无效，因为血液中的高水平毒素会导致死亡。 我们的研究计划集中在炭疽毒素抑制剂的开发上，炭疽毒素是由细菌分泌的三种蛋白质的组合。 保护性抗原（PA）结合受体并被蛋白酶切割，允许细胞相关的PA 63片段的七聚化。 七聚化允许酶毒素组分、水肿因子（EF）和致死因子（LF）的结合，并触发这些复合物的内吞作用。 内体的酸性环境导致酶蛋白易位到胞质溶胶中，在胞质溶胶中它们发挥其毒性作用。 EF是一种腺苷酸环化酶，通过多种机制损害先天免疫应答。 LF是一种引起巨噬细胞溶解的蛋白酶，导致休克样症状和死亡。 炭疽毒素受体（ATR）的发现将有助于抑制炭疽毒素作用的分子的开发。 我们将使用两种方法来分离PA-ATR相互作用的抑制剂，并测试这些抑制剂在体外阻断中毒过程的能力。 在第一种方法中，我们将选择结合ATR的肽，然后将该肽的多个拷贝连接到聚合物骨架上。 预测所得多价化合物以比单独的肽更高的亲和力结合ATR，并防止PA与细胞结合。 第二种方法是基于我们先前的观察，即ATR的可溶性片段可以在体外保护细胞免受毒素的侵害。 我们假设，一个突变片段，结合PA比野生型亲和力将是一个更有效的抑制剂。 我们将通过连续几轮的随机突变、选择和重组来分离该突变体。这些化合物可以延长炭疽病被成功治疗的时间。

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.