A SCREEN FOR SMALL MOLECULE INHIBITORS OF SHIGA TOXIN AND RICIN ACTIVITY

性状（申请人提供）：滋贺毒素（Stx）和蓖麻毒素，由某些E.大肠杆菌菌株和植物蓖麻毒素分别是人类疾病的重要原因，并且是生物战的潜在制剂。接触这些毒素的人的治疗选择有限。特别是，抗生素在蓖麻毒素暴露后没有作用，并且在暴露于产Stx的E.杆菌类似地，对于它们的失活，也没有特异性治疗剂来抑制由这些毒素引起的细胞死亡。我们的实验室最近进行了一项筛选小分子，抑制滋贺，蓖麻毒素和霍乱毒素的细胞内运输。我们已经确定了几种化合物，阻止这些毒素在宿主细胞内的运输。这些化合物是毒素转运途径的有用探针，并且一些可能具有作为治疗剂的潜力。与上述筛选（其是基于细胞的测定并靶向毒素转运）相结合，我们开发了用于毒素酶活性的生物化学测定。该测定具有高度灵敏度和重现性，并已适应高通量格式。通过分子库探针生产中心网络（MLPCN），我们建议筛选多达200，000种化合物，以确定其抑制Stx和蓖麻毒素酶活性的能力。将在二次试验中验证这些化合物中的每一种，然后对其效价、特异性、细胞毒性、可逆性和对相关毒素的影响进行表征。在未来的研究中，与南方研究所合作，具有最大治疗潜力的化合物将受到结构-活性关系（SAR）指导的效力，特异性，缺乏毒性和药物性质的优化。这些将在毒素介导疾病的动物模型中进行测试。公共卫生相关性：这项工作对公共卫生有重大影响。Stx每年在美国引起数十万例结肠炎，并导致数千人死亡。如果有意将其作为生物武器释放，这些毒素可造成严重的发病率和死亡率。我们建议识别和优化这些化合物作为可用于预防或治疗暴露于这些毒素的个体的第一种药物。

DESCRIPTION (provided by applicant): Shiga toxin (Stx) and ricin, produced by certain E. coli strains and the plant Ricin communicus, respectively, are important causes of human disease and are potential agents of biowarfare. Therapeutic options for persons exposed to these toxins are limited. In particular, antibiotics have no role following ricin exposure and are contraindicated in patients exposed to Stx-producing E. coli. Similarly, there is no specific therapeutics for their inactivation, nor for inhibition of the cell death caused by these toxins. Our laboratory has recently undertaken a screen for small molecules that inhibit intracellular transport of shiga, ricin, and cholera toxins. We have identified several compounds that block transport of these toxins within host cells. These compounds are useful probes of the toxin transport pathway and some may hold potential as therapeutic agents. In tandem with the above screen, which is a cell-based assay and targets toxin transport, we have developed a biochemical assay for toxin enzymatic activity. This assay is highly sensitive and reproducible and has been adapted to high-throughput format. Through the Molecular Libraries Probe Production Centers Network (MLPCN), we propose to screen up to 200,000 compounds for their ability to inhibit Stx and ricin enzymatic activity. Each of these compounds will be verified in a secondary assay then characterized with respect to their potency, specificity, cytotoxicity, reversibility, and effect on related toxins. In future studies, in collaboration with Southern Research Institutes, compounds with greatest therapeutic potential will be subjected to structure-activity relationship (SAR)-guided optimization of potency, specificity, lack of toxicity, and drug-like properties. These will be tested in an animal model of toxin-mediated disease. PUBLIC HEALTH RELEVANCE: This work has major implications for public health. Stx causes several hundred thousand cases of colitis in the US annually and accounts for thousands of deaths. In the event of their intentional release as bioweapons, these toxins could significance morbidity and mortality. We propose to identify and optimize as these compounds as the first drugs that could be used to prevent or treat individuals exposed to these toxins.