新型细菌成孔毒素抑制剂的设计、合成与活性评价

With the insurgence of drug-resistant super bugs, bacterial infection has become once again one of the most important health problems worldwide in recent years. Consequently, studies directed at the development of new therapeutic strategies against bacterial infections have been immerging as a renascent subject. Many infectious bacteria cause diseases through the secretion of so-called "pore-forming toxins".As the major virulent factors of these bacteria, pore-forming toxins have become a very attracting target for the development of novel antibacterial strategies. It has been further demonstrated that some of these toxins, such as aerolysin, CAMP factor and many others, bind to glycosylphosphatidylinositol (GPI) anchors present on the host cell surface to assist their formation of porous oligomers and then spontaneous insertion of the porous oligomers into the cell membrane to breach the cell permeability barrier, trigger adverse material exchanges, and finally cause cell death. As a result, a potential strategy for the treatment of related diseases is to develop new GPI-based inhibitors to prohibit the interactions between the pore-forming toxins and GPIs or GPI-anchored proteins on the host cell surface. This research project aims at the design and synthesis of a series of structurally well-defined GPI analogs as potential pore-forming toxin inhibitors and evaluation of their activities to inhibit the interaction between GPI anchors and pore-forming toxins, so as to find toxin inhibitors useful for the treatment of related diseases. In the meantime, new analytical methods will be developed for high throughput assays of toxin inhibitors using fluorescence-tagged GPI anchors as probes.Further more, the activities of these synthetic GPI analogs to inhibit GPI-toxin binding, inhibit the pore formation of toxins in lipid membrane and inhibit toxin-induced cytotoxicity will be investigated by using a GPI-decorated cell-mimic liposome model,by titration of toxin-induced hemolysis,and by protection of mice against bacterial infections and bacterial toxins.This research will not only yield novel pore-forming toxin inhibitors or promising lead compounds worthy further investigations but also provide with the structure-activity relationships (SARs) of GPI analogs as toxin inhibitors to guide further optimization of the inhibitor design. The results will have a major impact on antibacterial research.

近年来，随着耐药菌株的不断涌现，细菌感染再次成为人类健康的严重威胁，针对细菌感染的新药研发成为国际热点之一。许多细菌致病是由其分泌的成孔毒素所引起，成孔毒素因此成为研发抗菌新药的重要靶点。研究表明，气单胞菌溶素和CAMP因子等成孔毒素通过与宿主细胞表面的糖基磷脂酰肌醇（GPI）结合助其聚集成孔状低聚物，并自发地插入细胞膜中形成孔穴，从而破坏细胞的渗透屏障，导致反向离子交换和其他致命反应，最终杀死宿主细胞。因此，一种潜在抗菌新策略是发展基于GPI结构的新型毒素抑制剂来阻断成孔毒素与宿主细胞表面GPI的结合。本申请项目拟设计合成一系列GPI衍生物，探究其对成孔毒素与GPI结合的影响，发现成孔毒素抑制剂；同时发展一种以GPI荧光探针为基础的高通量筛选方法，用以评价毒素抑制剂的活性；此外，通过GPI修饰的脂质体模型、体外溶细胞、动物体内保护等实验，评价所设计毒素抑制剂的抗毒活性，并阐明其构效关系。

许多细菌致病是由其分泌的成孔毒素所引起，而宿主细胞表面的糖基磷脂酰肌醇（GPI）与成孔毒素结合助其在细胞膜成孔而产生感染疾病。因此，发展基于GPI结构的新型毒素抑制剂来阻断成孔毒素与宿主细胞表面的GPI的结合是一种潜在的抗菌新策略。本项目设计合成了一系列具有不同糖链长度且不同功能化糖基修饰的GPI衍生物，用于探究其对成孔毒素与GPI结合的影响；设计合成了3种基于BODIPY荧光标签的GPI荧光探针，用于毒素抑制剂的活性的高通量筛选；制备了GPI修饰的脂质体模型，用于毒素抑制剂的抗毒活性评价。同时，本项目发展了一种基于代谢工程技术来检测细胞表面GPI锚定蛋白的方法，能够实现了对细胞表面GPI锚和GPI锚定蛋白的标记，进而研究其生物功能。此外，本项目还探索研究了GPI核心二糖生物合成相关的生物酶蛋白，发现了源自C. diphtheriae的MshA具有Ｎ-乙酰葡萄糖基转移酶的活性，而源自C. glutamicum磷酸酶ImpC具有脱磷酸的活性。上述研究工作不仅为今后继续开展新型基于GPI结构的毒素抑制剂的筛选奠定了物质基础，同时也为研究细胞表面GPI分子的标记与功能研究提供了有效的分析手段以及GPI的生物酶法合成奠定了研究基础。