Blocking Cellular Intoxication by Bacterial Toxins

阻止细菌毒素引起的细胞中毒

• 批准号: 6561325
• 负责人: Steven R. Blanke
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561325
• 关键词: Clostridium botulinum; acidity /alkalinity; biotechnology; bioterrorism /chemical warfare; botulinum toxins; disulfide bond; inhibitor /antagonist; membrane permeability; mutant; protein engineering; protein protein interaction; recombinant proteins

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.

描述（由申请人提供）：BoNT 因其极高的效力和杀伤力以及易于生产和运输而成为一种危险的生物恐怖主义威胁。如果不及时治疗，肉毒毒素中毒可能会发展为弛缓性麻痹，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，长期研究目标是开发一种新型疗法，可用于 BoNT 中毒的个体。如本申请中所描述的，该策略将是开发一种毒素 BoNT/A 形式的显性失活突变体，该突变体将与野生型毒素的复合物相互作用并使复合物失活。 BoNT/A 由两个定义的片段组成。 H 链促进第二个毒素片段（L 链）转运到靶细胞中。该策略基于这样的假设：BoNT/AL L 链的转运是细胞中毒机制中的重要步骤，是由 pH 触发的解折叠和毒素寡聚复合物的膜插入介导的。实验和计算方法将用于开发 H 链介导的 L 链膜转运机制的模型。该模型将通过以预测干扰膜转运的方式改变 H 链的氨基酸序列来测试。具体而言，将在 H 链内设计分子内二硫键，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，带正电荷的氨基酸将被引入整个H链，预计这不利于BoNT/A H链的膜插入。 H链的野生型和突变体形式将被表达为重组蛋白，并且将在野生型毒素存在的情况下测试每个突变体的显性失活抑制活性。同时，对易位重要的结构-功能关系将被确定为未来设计基于显性失活的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失活的 BoNT/A 突变体，这些突变体将使用体外模型系统阻断野生型毒素的作用。这项研究的结果将为这些新型抑制剂的未来开发和使用已建立的动物模型进行体内测试奠定基础和合理性。