Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design
Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design
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DOI：
10.1073/pnas.0507488102
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发表时间：
2005-10-18
影响因子：
11.1
通讯作者：
Bezrukov, SM
中科院分区：
文献类型：
--
作者：
Karginov, VA;Nestorovich, EM;Bezrukov, SM
炭疽杆菌分泌三种息肉：保护性抗原（PA）、致死因子（LF）和水肿因子（EF），它们在哺乳动物细胞表面相互作用形成毒性复合物。 LF 和 EF 是针对细胞质内底物的酶； PA 提供七聚孔以促进 LF 和 EF 转运到细胞质中。除了暴露后立即使用抗生素外，目前还没有批准的有效治疗吸入性炭疽的方法。在这里，我们展示了一种禁用毒素的方法：通过合理设计的低分子量化合物对 PA 孔进行高亲和力堵塞，防止 LF 和 EF 进入细胞。在PA孔的七重对称性和主要负电荷的指导下，我们合成了七重对称性的小环状分子，即经过化学修饰以添加七个正电荷的β-环糊精。通过通道重建和高分辨率电导记录，我们发现 per-6-(3-氨基丙硫基)-β-环糊精与 PA 孔腔强烈相互作用，在亚纳摩尔浓度（0.1 M KCl 中）阻断 PA 诱导的运输。该化合物保护 RAW 264.7 小鼠巨噬细胞免受炭疽致死毒素 (= PA + LF) 的细胞毒性。更重要的是，它完全保护了高度易感的 Fischer F344 大鼠免受致命毒素的侵害。我们预计这种方法将作为结构导向药物发现计划的基础，以寻找新的有效的炭疽治疗方法。
Bacillus anthracis secretes three polypepticles: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, beta-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-beta-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCl). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin (= PA + LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.