Salmonella-specific therapeutics

项目摘要 非伤寒沙门氏菌病是美国和全球最重要的食源性疾病之一。我们 最近使用高通量遗传筛选来鉴定沙门氏菌fra基因座，其突变导致 老鼠的适应能力极度减弱。然后，我们确定了fra基因座编码五个基因， 果糖-天冬酰胺（F-Asn）的吸收和利用：fraR、fraB、fraD、fraA和fraE。FRA位点被发现 仅在非伤寒沙门氏菌血清型、少数柠檬酸杆菌属和克雷伯氏菌属分离株以及少数 梭状芽孢杆菌因此，用新的抗微生物剂靶向沙门氏菌中该位点的产物， 使正常的微生物群基本上完好无损。我们对衰减机制的表征表明， fraB中的突变导致FraB底物- 6-磷酸果糖-天冬氨酸（6-P-F-Asp）- 对细胞有毒的物质。我们提出了高通量筛选（HTS）与三种不同的测定，以确定小 FraB的分子抑制剂，FraB是一种将6-P-F-Asp转化为天冬氨酸和葡萄糖-6-P（Glc-6-P）的脱糖酶。一 一种测定法在分光光度测定法中利用纯化的FraB酶，而另一种是基于生长的测定法 利用减毒活沙门氏菌和沙门氏菌对照。我们测试了生物化学和基于细胞的测定， 在哈佛的ICCB-Longwood设备上，发现它们是简单和稳健的，Z' ≥0.9和≥0.8， 分别我们建议使用这两种检测方法来鉴定FraB抑制剂，以筛选多达50万个 在ICCB-Longwood设施的化合物。在第三个试验中，我们将使用基于计算机结构的虚拟 从NCI数据库中筛选约25万种化合物。ICCB和朗伍德两个屏幕上的点击率 计算机屏幕将在我们的研究所再次进行测试。第二次独立确认 将利用基于质谱的测定直接测量6-P-F-Asp的积累，6-P-F-Asp是FraB的底物， 在活细胞中。命中将进一步表征其IC 50、IC 90、Ki和特异性。计算 化学将被用来更好地了解FraB抑制剂的化学概况，并促进 定量构效关系（QSAR）研究。此外，为了获得效力的结构基础， 命中，我们将使用X射线晶体学，以确定原子分辨率的结构FraB与和没有 选择抑制剂。这些目标的成功实现将有助于确定打击目标， 表征，关键的预处理，为铅优化和进步，以迫切需要的窄谱 治疗非伤寒沙门氏菌病。窄谱抗生素将具有两个关键优势：（i）限制 由正常微生物群破坏引起的副作用，以及（ii）避免选择抗微生物剂 正常微生物群中的抵抗力。我们设想未来的鸡尾酒物种特异性药物， 用于治疗人类腹泻而不破坏健康的微生物群。一种药物， 本提案中确定的命中结果将是这一混合物的一个组成部分。

Project Summary Non-typhoidal salmonellosis is one of the most significant food-borne diseases in the U.S. and globally. We recently used high-throughput genetic screening to identify the Salmonella fra locus, whose mutation causes extreme attenuation of fitness in mice. We then determined that the fra locus encodes five genes involved with the uptake and utilization of fructose-asparagine (F-Asn): fraR, fraB, fraD, fraA, and fraE. The fra locus is found only in the non-typhoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium. Thus, targeting the products of this locus in Salmonella with novel antimicrobials is expected to leave the normal microbiota largely intact. Our characterization of the mechanism of attenuation revealed that mutations in fraB cause an accumulation of the FraB substrate – 6-phosphofructose-asparate (6-P-F-Asp) – that is toxic to cells. We propose high-throughput screening (HTS) with three different assays to identify small molecule inhibitors of FraB, a deglycase that converts 6-P-F-Asp to aspartate and glucose-6-P (Glc-6-P). One assay utilizes purified FraB enzyme in a spectrophotometric assay, while another is a growth-based assay utilizing a live-attenuated Salmonella and a ∆fra control. We tested the biochemical and cell-based assays at the ICCB-Longwood facility at Harvard, and found them to be simple and robust with Z' ≥0.9 and ≥0.8, respectively. We propose to identify FraB inhibitors using these two assays to screen up to 500,000 compounds at the ICCB-Longwood facility. In the third assay, we will use in silico structure-based virtual screening of ~250,000 compounds from the NCI database. The hits from both of the ICCB-Longwood screens and the computational screens will be tested again at our home institution. A second independent confirmation will utilize a mass spectrometry-based assay to directly measure build-up of 6-P-F-Asp, the substrate of FraB, in live cells. Hits will be characterized further with regard to their IC50, IC90, Ki, and specificity. Computational chemistry will be employed to better understand the chemical profile of FraB inhibitors, and facilitate quantitative structure-activity relationship (QSAR) studies. Moreover, to gain a structural basis for the potency of hits, we will use X-ray crystallography to determine the atomic-resolution structure of FraB with and without select inhibitors. Successful completion of these aims is expected to facilitate hit identification and characterization, key pre-requisites for lead optimization and advancement to a much needed narrow-spectrum therapeutic for non-typhoidal salmonellosis. Narrow-spectrum antibiotics will have two key advantages: (i) limit the side effects caused by disruption of the normal microbiota, and (ii) avoid selecting for antimicrobial resistance among the normal microbiota. We envision a future cocktail of species-specific drugs that could be used to treat cases of human diarrhea without disruption of the healthy microbiota. A drug that ultimately results from the hits identified in this proposal would be one component of this cocktail.