Salmonella-specific therapeutics

项目摘要 非伤寒沙门氏菌病是美国和全球最严重的食源性疾病之一。我们 最近使用高通量基因筛查来鉴定沙门氏菌fra基因座，其突变导致 使小鼠的体能极度衰弱。然后，我们确定了fra基因座编码了五个与 果糖-天冬酰胺(F-ASN)的吸收和利用：FRAR、FRAB、FRAD、FRAA和FRAE。找到了fra基因的位置 仅在非伤寒沙门氏菌血清中，少数柠檬酸杆菌和克雷伯氏菌分离株，以及少数几种 梭状芽胞杆菌因此，在沙门氏菌中以该基因座的产物为靶点的新型抗菌剂有望 让正常的微生物区系基本保持不变。我们对衰减机制的描述揭示了 FRAb突变导致FRAb底物-6-磷酸果糖-天冬氨酸(6-P-F-Asp)积聚- 这对细胞是有毒的。我们建议采用高通量筛查(HTS)和三种不同的检测方法来识别 FRAb的分子抑制剂，是一种将6-P-F-Asp转化为天冬氨酸和葡萄糖-6-P(GLC-6-P)的脱糖体。一 在分光光度分析中使用纯化的FRAb酶，而另一种是基于生长的分析 使用减毒活沙门氏菌和∆FRA对照。我们测试了生化和基于细胞的分析， 位于哈佛的ICCB-Longwood设施，发现它们在Z‘≥0.9和≥0.8中简单而健壮， 分别进行了分析。我们建议使用这两种方法来鉴定FRAb抑制剂，以筛选多达500,000 ICCB-Longwood工厂的化合物。在第三个测试中，我们将使用基于硅胶结构的虚拟 从NCI数据库中筛选出约250,000种化合物。来自ICCB-Longwood两部银幕的热门影片 计算屏幕将在我们的本土机构再次进行测试。第二次独立确认 将利用基于质谱学的分析方法直接测量FRAB底物6-P-F-Asp的积聚， 在活细胞中。HITS将在IC50、IC90、KI和特异性方面进一步表征。计算型 将利用化学来更好地了解FRAb抑制剂的化学特征，并促进 定量构效关系(QSAR)研究。此外，为了获得效力的结构性基础 对于HITS，我们将使用X射线结晶学来确定有无FRAB的原子分辨结构 选择抑制剂。这些目标的成功完成预计将有助于命中识别和 表征、Lead优化的关键前提条件以及向急需的窄谱方向发展 治疗非伤寒沙门氏菌病。窄谱抗生素将有两个主要优势：(I)限制 破坏正常微生物区系造成的副作用，以及(Ii)避免选择抗菌剂 在正常微生物区系中产生抗药性。我们设想了一种未来的特定物种药物的鸡尾酒，它可能是 用于治疗人类腹泻病例，而不破坏健康的微生物区系。一种药物，最终 这项提案中确定的点击率结果将是这杯鸡尾酒的一个组成部分。

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.