Blocking Cellular Intoxication by Bacterial Toxins

描述（由申请人提供）：bont是一种危险的生物恐怖主义威胁，因为它们具有极强的效力和杀伤力，而且易于生产和运输。如果不及时治疗，BoNTs中毒可发展为弛缓性麻痹，并因呼吸衰竭而死亡。然而，及时的接触后干预可以限制循环毒素的影响。总的来说，长期的研究目标是产生一种新的治疗方法，可以给那些被BoNT中毒的人。如本申请中所述，该策略将是开发一种毒素BoNT/A的显性阴性突变体，这种突变体将与野生型毒素的复合物相互作用并使其失活。BoNT/A由两个定义的片段组成。H链促进第二个毒素片段，即L链，进入靶细胞。该策略基于这样的假设，即BoNT/A L链的运输是细胞中毒机制的重要步骤，是由ph触发的毒素寡聚物的展开和膜插入介导的。实验和计算方法将用于建立H链介导的L链膜运输机制的模型。该模型将通过改变H链的氨基酸序列来测试，以一种预测会干扰膜运输的方式。具体来说，分子内二硫键将在H链内进行设计，以限制由于pH触发的构象变化而导致的多肽主链的移动。此外，带正电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，并且在野生型毒素存在的情况下，将对每个突变体进行显性阴性抑制活性测试。同时，对于易位重要的结构-功能关系将被确定为未来设计显性阴性抑制剂的重要先决条件。这项工作的一个里程碑将是鉴定一个或多个显性阴性BoNT/A突变体，这些突变体将在体外模型系统中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理由。

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.