A Screen for Small Molecule Compounds that Inhibit Bacterial Toxins

描述(申请人提供)：志贺毒素(STX)、霍乱毒素(CTX)和蓖麻毒素是人类疾病的重要媒介，是潜在的生物武器制剂。这些毒素遵循复杂的细胞内途径，以杀死敏感细胞。在与细胞表面分子结合后，这些毒素被内化，并以逆行的方式通过高尔基体进入内质网。从内质网腔，毒素必须进入细胞质，在那里它们进行细胞毒活动。最近发现的这一途径可能代表了一种循环和细胞质量控制机制，但迄今尚未得到很好的描述。逆行转运途径被不同来源的毒素所利用，是治疗干预的一个有吸引力的靶点。然而，逆行交通的机制和涉及的宿主分子在很大程度上是未知的。我们正在使用STX敏感性抑制剂的小分子筛查作为一种工具来剖析毒素转运途径的成分。我们已经开发了几种方法来检测抑制化合物对逆行和正向(分泌)运输的影响。这项提议的目标是确定另外几种化合物，其中包括在毒素从质膜运输到内质网管腔的过程中在多个位置抑制毒素的化合物。我们的检测是基于荧光素酶活性作为毒素抗性的报告。用表达荧光素酶的腺病毒载体转导细胞，该荧光素酶已被蛋白酶体修饰为可迅速降解。暴露于STX会抑制蛋白质合成，导致荧光素酶含量显著降低。抑制STX转运或活性的化合物会导致荧光素酶活性的恢复，并很容易在发光计中检测到。为了排除假阳性，加入了一个由放线菌亚胺孵育而不是STX孵育组成的反筛选。筛选和反筛选的实用性在ICCB设施中得到了验证，在那里我们发现z和z‘得分为&gt；0.6，并展示了排除对荧光素酶活性有非特异性影响的化合物的能力。基于我们前一轮的筛选，我们预计将从10万个分子中鉴定出至少50种抑制性化合物。我们将通过测定它们的效力、特异性、毒性、可逆性以及对它们在体外毒素转运的作用进行初步评估，对这些化合物进行后续研究。根据这项分析的结果，这些化合物将被优先纳入研究，以确定毒素抑制的位置和抑制化合物的细胞内靶标。这里提出的调查将确定有效和特定的毒素贩运抑制物。这些化合物将被用作探针，以识别参与毒素运输不同阶段的运输途径和宿主分子。在未来的实验中，其中一些化合物将被优化为潜在的治疗剂。志贺毒素、霍乱毒素和蓖麻毒素是人类疾病的重要病原体，被认为是潜在的生物武器制剂。这些毒素和其他毒素遵循复杂的细胞内途径，以杀死敏感细胞。这一转运途径是治疗干预的一个有吸引力的靶点。我们正在使用小分子筛选志贺毒素作用的抑制剂。对志贺毒素有活性的化合物将被检测其抑制蓖麻毒素和霍乱毒素的能力。然后将根据它们的效力、毒性、可逆性和作用机制来表征这些化合物。得到的化合物将是剖析毒素和囊泡运输途径的宝贵工具。在最终目标中，将识别抑制分子的宿主靶标。从长远来看，抑制毒素转运并在体外表现出最低毒性的化合物将被探索为潜在的治疗剂。

DESCRIPTION (provided by applicant): Shiga toxin (Stx), cholera toxin (Ctx) and ricin are important mediators of human disease and are potential agents of biowarfare. These toxins follow a complex intracellular pathway in order to kill susceptible cells. After binding to cell surface molecules, these toxins are internalized and trafficked in retrograde fashion through the Golgi to the endoplasmic reticulum (ER). From the ER lumen, the toxins must gain access to the cytoplasm, where they carry out their cytotoxic activities. This recently discovered pathway likely represents a recycling and cellular quality control mechanism that has as yet been poorly characterized. The retrograde transport pathway, exploited by toxins of widely different origin, is an attractive target for therapeutic intervention. However, the mechanisms of retrograde traffic and the host molecules involved are largely unknown. We are employing a small molecule screen for inhibitors of Stx susceptibility as a tool to dissect the components of toxin transport pathways. We have developed several assays to examine the effects of inhibitory compounds on retrograde and forward (secretory) transport. The goal of this proposal is to identify several more compounds that, among them, inhibit toxins at multiple sites in their transport from plasma membrane to ER lumen. Our assay is based on luciferase activity as a reporter for toxin resistance. Cells are transduced with an adenoviral construct expressing luciferase that has been modified to be degraded rapidly by the proteasome. Exposure to Stx, which inhibits protein synthesis, results in markedly diminished luciferase content. Compounds that inhibit Stx transport or activity result in a rescue of the luciferase activity and are readily detected in a luminometer. A counterscreen, consisting of incubation with cycloheximide rather than Stx, was incorporated to exclude false-positive hits. The utility of the screen and counterscreen were validated at the ICCB facility, where we found z and z' scores to be > 0.6 and demonstrated the ability to exclude compounds with nonspecific effects on luciferase activity. Based on our previous round of screening we anticipate identifying at least 50 inhibitory compounds from a screen of 100,000 molecules. We will follow up on these compounds by determining their potency, specificity, toxicity, reversibility, and a preliminary assessment of their effect on toxin trafficking in vitro. Based on the results of this analysis, the compounds will be prioritized for their inclusion into studies that will identify the site of toxin inhibition and the intracellular targets of inhibitory compounds. Investigations proposed here will identify potent and specific inhibitors of toxin trafficking. These compounds will be used as probes to identify trafficking pathways and host molecules involved in various stages of toxin transport. In future experiments, some of these compounds will be optimized for potential use as therapeutic agents. Shiga toxin, cholera toxin and ricin are important agents of human disease and are recognized as potential agents of biowarfare. These and other toxins follow a complex intracellular pathway in order to kill susceptible cells. This transport pathway is an attractive target for therapeutic intervention. We are employing a small molecule screen for inhibitors of shiga toxin action. Compounds with activity against shiga toxin will be examined for their ability to inhibit ricin and cholera toxin. The compounds will then be characterized with respect to their potency, toxicity, reversibility, and mechanism of action. The resulting compounds will be valuable tools to dissect toxin and vesicle trafficking pathways. In the final aim, the host targets of inhibitory molecules will be identified. Over the long term, compounds that inhibit toxin trafficking and display minimal toxicity in vitro will be explored as potential therapeutic agents.