Blocking Cellular Intoxication by Bacterial Toxins

描述(申请人提供)：BoNTs是一种危险的生物恐怖主义威胁，因为它们具有极高的效力和杀伤力，以及它们易于生产和运输。如果不治疗，BoNTs的中毒可能会进展为软性瘫痪，并因呼吸衰竭而死亡。然而，及时的暴露后干预可以限制循环毒素的影响。总体而言，长期研究的目标是产生一种新的治疗方法，可以用来治疗被BONT中毒的个人。如本申请所述，该策略将开发一种形式的毒素BONT/A的显性-负性突变体，该突变体将与野生型毒素的复合体相互作用并使其失活。BONT/A由两个定义的片段组成。H链促进了第二个毒素片段L链向靶细胞的运输。这一策略是基于这样的假设，即作为细胞中毒机制中必不可少的一步，BONT/A L链的运输是由pH触发的毒素低聚物的展开和膜插入介导的。采用实验和计算相结合的方法，建立了H链介导的L链膜转运机制的模型。这一模型将通过改变H链的氨基酸序列来测试，预计这种方式会干扰膜的运输。具体地说，分子内二硫键将被设计在H链内，以限制由于pH引发的构象变化而导致的多肽主干的移动。此外，带正电荷的氨基酸将被引入整个H链，这将不利于BONT/A H链的膜插入。野生型和突变型的H链将以重组蛋白的形式表达，每个突变体将在野生型毒素存在的情况下进行显性负抑制活性测试。同时，对易位至关重要的结构-功能关系将被确定为显性-负性基础抑制剂功能设计的重要前提。这项工作的一个里程碑将是利用体外模型系统鉴定一个或多个显性阴性的BONT/A突变体，这些突变体将阻断野生型毒素的作用。这项研究的结果将为未来开发和使用已建立的动物模型进行这些新型抑制剂的体内测试奠定基础和合理性。

DESCRIPTION (provided by applicant): BoNTs are a dangerous bioterrorism threat due to their extreme potency and lethality, as well as their ease of production and transport. If untreated, poisoning by the BoNTs can progress to flaccid paralysis and death due to respiratory failure. However, timely post-exposure intervention can limit the effects of the circulating toxin. The overall, long-term research objective is to generate a novel class of therapeutics that can be administered to individuals who have been poisoned by BoNT. The strategy, as described in this application, will be to develop dominant-negative mutants of one form of the toxin, BoNT/A, which will interact with and inactivate complexes of wild type toxin. BoNT/A is composed of two defined fragments. The H chain facilitates the transport of a second toxin fragment, the L chain, into target cells. The strategy is based on the hypothesis that transport of the BoNT/A L chain, which is an essential step in the cellular intoxication mechanism, is mediated by pH-triggered unfolding and membrane insertion of toxin oligomeric complexes. Experimental and computational approaches will be used to develop a model for the mechanism of H chain-mediated membrane transport of the L chain. This model will be tested by altering the amino acid sequence of the H chain in a manner predicted to interfere with membrane transport. Specifically, intramolecular disulfide linkages will be engineered within the H chain to limit movement of the polypeptide backbone as a result of pH triggered conformational changes. Moreover, positively charged amino acids will be introduced throughout the H chain, which would be predicted to be unfavorable for membrane insertion of the BoNT/A H chain. Wild type and mutant forms of the H chain will be expressed as recombinant proteins, and each mutant will be tested for dominant-negative inhibitory activity in the presence of wild type toxin. Simultaneously, structure-function relationships important for translocation will be identified as an important prerequisite for fu1ure design of dominant-negative based inhibitors. A milestone of this work will be the identification of one or more dominant-negative BoNT/A mutants that will block action of wild type toxin using in vitro model systems. The results from this research will establish the groundwork and justification for future development and in vivo testing of these novel inhibitors using established animal models.