A Small Molecule Approach Toward Understanding Bacterial Toxin Transport

了解细菌毒素运输的小分子方法

• 批准号: 7615821
• 负责人: Jose Bernardo Saenz
• 金额: $ 2.79万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7615821
• 关键词: ADP-Ribosylation Factor 1; Bacterial Toxins; Binding; Biochemical; Biological Assay; Bioterrorism; Brefeldin A; Bypass; Cells; Cholera Toxin; Complex; Cytosol; Destinations; Disease; Endocytosis; Endoplasmic Reticulum; Endosomes; Equilibrium; Genetic; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Homeostasis; Human; Immunofluorescence Immunologic; Intoxication; Intracellular Transport; Libraries; Luciferases; Maps; Mediating; Membrane; Methods; Monitor; Organelles; Pathway interactions; Phosphotransferases; Plants; Protein Isoforms; Protein Kinase; Protein Synthesis Inhibition; Radioactive; Ricin; Role; Route; Shiga Toxin; Site; Small Interfering RNA; Specificity; Staging; Structure-Activity Relationship; System; Targeted Toxins; Therapeutic; Toxin; Translating; Vesicle; Virulence; base; cytotoxicity; high throughput screening; human disease; inhibitor/antagonist; kinase inhibitor; knock-down; mutant; novel; pharmacophore; retrograde transport; small molecule; small molecule libraries; tool; trafficking

DESCRIPTION (provided by applicant): Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via complex intracellular routes. Though diverse in their intracellular targets, a common and essential step in their virulence is the ability to reach the cytosol, where most toxins exert their enzymatic effects. The bacterial products Stx and Ctx, as well as the plant toxin ricin, traffick in a retrograde fashion from endosomes to the endoplasmic reticulum via the Golgi while bypassing lysosomal degradation. It has become increasingly apparent that this retrograde trafficking pathway is not unique to bacterial toxins. Rather, bacterial toxins are believed to hijack existing host transport pathways. The sequential retrograde progression utilized by these toxins has translated into a unique system for probing host endocytic mechanisms and have enhanced our understanding of retrograde transport. In an effort to dissect and inhibit the stepwise trafficking of Stx, we have developed an alternative approach toward inhibiting toxin transport. Previous genetic approaches aimed at identifying components essential to toxin trafficking have proven inconsistent and revealed the complexity underlying these pathways. As a result, we have applied a highly sensitive, high-throughput luciferase-based assay to screen a library of small molecule compounds for their ability to block inhibition of protein synthesis. From an initial screen, we have identified various compounds that strongly protect against Shiga intoxication. One novel compound, B06, produces a brefeldin A-like dispersal of the Golgi, and preliminary evidence suggests that it could serve as a useful tool in elucidating ARF1-mediated transport through the Golgi apparatus. In addition, we hope to explore the role of kinases in toxin trafficking. Though there is evidence for the role of kinases in toxin endocytosis, their functions in intracellular toxin trafficking remain unclear. A novel kinome siRNA library will be screened by the luciferase-based assay to identify kinases essential to Stx transport. More importantly, the identification of kinases involved at various stages along the Stx trafficking pathway will further our understanding of their functions in mediating intracellular transport. Identification of kinases involved at distinct steps in toxin trafficking will equally provide therapeutic possibilities against toxin-mediated diseases. Bacterial and plant toxins are significant agents of human disease and potential vehicles for bioterrorism. Our high-throughput assay underscores the therapeutic utility of a small molecule approach for targeting toxin-mediated diseases.

描述（由申请人提供）：滋贺毒素（Stx）、霍乱毒素（Ctx）和植物毒素蓖麻毒素是通过复杂的细胞内途径到达其细胞内目的地的几种毒素。虽然它们的细胞内靶点不同，但它们毒力的一个共同和重要步骤是到达细胞溶质的能力，大多数毒素在细胞溶质中发挥其酶促作用。细菌产物Stx和Ctx，以及植物毒素蓖麻毒素，以逆行的方式从内体通过高尔基体运输到内质网，同时绕过溶酶体降解。越来越明显的是，这种逆行运输途径并不是细菌毒素所独有的。相反，细菌毒素被认为是劫持现有的宿主运输途径。这些毒素利用的连续逆行进展已经转化为探测宿主内吞机制的独特系统，并增强了我们对逆行转运的理解。在努力剖析和抑制Stx的逐步贩运，我们已经开发出一种替代的方法来抑制毒素运输。以前的遗传学方法，旨在确定毒素贩运必不可少的组件已被证明是不一致的，并揭示了这些途径的复杂性。因此，我们应用了一种高灵敏度、高通量的基于酶的测定来筛选小分子化合物库，以确定其阻断蛋白质合成抑制的能力。从最初的筛选，我们已经确定了各种化合物，强烈防止滋贺中毒。一种新的化合物，B06，产生一个布雷菲德菌素A样分散的高尔基体，初步证据表明，它可以作为一个有用的工具，在阐明ARF1介导的运输通过高尔基体。此外，我们希望探索激酶在毒素运输中的作用。虽然有证据表明激酶在毒素内吞作用中的作用，但它们在细胞内毒素运输中的功能仍不清楚。一个新的激酶组siRNA文库将通过基于激酶的测定来筛选，以鉴定Stx转运所必需的激酶。更重要的是，激酶参与沿着的Stx运输途径的各个阶段的鉴定将进一步我们了解他们的功能，介导细胞内运输。在毒素运输的不同步骤参与激酶的鉴定将同样提供针对毒素介导的疾病的治疗可能性。细菌和植物毒素是人类疾病的重要病原体，也是生物恐怖主义的潜在载体。我们的高通量测定强调了小分子方法用于靶向毒素介导的疾病的治疗效用。