Salmonella-specific therapeutics

沙门氏菌特异性疗法

• 批准号: 9764264
• 负责人: Brian M Ahmer
• 金额: $ 71.95万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764264
• 关键词: Adult; Adverse effects; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Apricot; Asparagine; Aspartate; Attenuated; Bacteria; Binding; Biochemical; Biological Assay; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Child; Citrobacter; Clostridium; Complex; Computer Simulation; Crystallization; Databases; Defect; Development; Diarrhea; Drug Targeting; Elderly; Enzymes; Food; Fos-Related Antigens; Foundations; Free Energy; Fructose; Future; Gastroenteritis; Genes; Genetic Screening; Glucose; Glucose-6-Phosphate; Goals; Growth; Home environment; Homology Modeling; Human; Immunocompromised Host; Institution; Klebsiella; Mammals; Mass Spectrum Analysis; Measures; Mediating; Metabolism; Methods; Mus; Mutation; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Predisposition; Quantitative Structure-Activity Relationship; Recombinants; Reporting; Resistance; Resistance development; Resolution; Salmonella; Salmonella Pathway; Salmonella enterica; Salmonella infections; Serotyping; Severities; Specificity; Structure; System; Testing; Therapeutic; United States; Vaccines; X-Ray Crystallography; antimicrobial; attenuation; base; computational chemistry; drug discovery; enzyme pathway; fitness; foodborne illness; foodborne infection; foodborne pathogen; gut microbiota; high throughput screening; indexing; inhibitor/antagonist; lead optimization; lead series; microbiota; mutant; non-typhoidal Salmonella; normal microbiota; novel; novel therapeutics; pathogen; pharmacokinetics and pharmacodynamics; screening; side effect; small molecule inhibitor; therapeutic target; uptake; virtual

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.

项目摘要 非细类藻类病是美国和全球最重要的食物传播疾病之一。我们 最近使用高通量遗传筛查来识别沙门氏菌FRA基因座，其突变引起 小鼠健身的极端衰减。然后，我们确定FRA基因座编码涉及的五个基因 果糖 - 天冬酰胺（F-ASN）的吸收和利用：Frar，Frab，Frad，Fraa和Frae。发现FRA基因座 只有在非细类沙门氏菌血清中 梭状芽胞杆菌。那是针对沙门氏菌中该基因座的产品的，预计将 留下正常的微生物群很大程度上完整。我们对衰减机制的表征表明 FRAB中的突变会导致FRAB底物的积累 - 6-磷酸果糖 - 叠蛋白（6-P-F-ASP） - 那对细胞有毒。我们建议使用三种不同测定的高通量筛选（HTS）来识别小 FRAB的分子抑制剂，一种将6-P-F-ASP转换为天冬氨酸和葡萄糖-6-P（GLC-6-P）的脱脂酶。一 测定在分光光度测定中利用纯化的包裹酶，而另一种是基于生长的测定法 使用活衰减的沙门氏菌和∆FRA控制。我们测试了生化和基于细胞的测定 哈佛大学的ICCB-longwood设施，发现它们很简单且健壮，≥0.9和≥0.8， 分别。我们建议使用这两种测定法鉴定包裹抑制剂，以筛选最多500,000 ICCB-longwood设施的化合物。在第三种测定中，我们将用于基于硅结构的虚拟 从NCI数据库筛选约25万种化合物。 ICCB-longwood屏幕的命中 并且计算屏幕将在我们的家庭机构再次进行测试。第二个独立确认 将利用基于质谱的测定法直接测量6-P-F-ASP的堆积，FRAB的底物， 在活细胞中。命中将进一步表征其IC50，IC90，KI和特异性。计算 化学将被雇用以更好地了解包裹抑制剂的化学特征，并促进 定量结构活性关系（QSAR）研究。此外，要获得效力的结构性基础 在命中中，我们将使用X射线晶体学来确定带有和不带有和不带有的原子分辨率结构 选择抑制剂。这些目标的成功完成有望促进命中识别和 表征，铅优化的关键先决条件以及急需的狭窄光谱的进步 非细类细构病的治疗性。狭窄的光谱抗生素将具有两个关键优势：（i）限制 正常菌群的破坏引起的副作用，（ii）避免选择抗菌剂 正常微生物群中的抗性。我们设想了未来的鸡尾酒，包括特定规格的药物 用于治疗人腹泻病例，而不会破坏健康的微生物群。最终的药物 该提案中确定的点击量的结果将是该鸡尾酒的一部分。