Salmonella-specific therapeutics

沙门氏菌特异性疗法

基本信息

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
项目类别：
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

项目摘要

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.

项目概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。最近使用高通量遗传筛选来鉴定沙门氏菌基因座，其突变导致然后我们确定 fra 基因座编码与小鼠适应性的极度减弱。果糖天冬酰胺 (F-Asn) 的摄取和利用：fraR、fraB、fraD、fraA 和 fraE 发现 fra 基因座。仅存在于非伤寒沙门氏菌血清型、少数柠檬酸杆菌和克雷伯氏菌分离株以及少数种因此，用新型抗菌剂靶向沙门氏菌中该基因座的产物有望实现。我们对减毒机制的表征表明，正常微生物群基本完好无损。 fraB 的突变导致 FraB 底物的积累 – 6-磷酸果糖-天冬氨酸 (6-P-F-Asp) – 我们建议通过三种不同的检测方法进行高通量筛选（HTS）来识别小分子。 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) 的去糖酶。一种测定在分光光度测定中使用纯化的 FraB 酶，而另一种测定是基于生长的测定我们利用减毒活沙门氏菌和 Δfra 对照进行了生化和细胞检测。哈佛大学的 ICCB-Longwood 设施，发现它们简单而稳健，Z' ≥0.9 和 ≥0.8，我们建议分别使用这两种检测方法来筛选多达 500,000 个 FraB 抑制剂。在 ICCB-Longwood 设施中的化合物在第三次测定中，我们将使用基于计算机结构的虚拟。从 NCI 数据库筛选约 250,000 种化合物来自 ICCB-Longwood 筛选的结果。计算屏幕将在我们所在的机构再次进行第二次独立确认。将利用基于质谱的测定法直接测量 FraB 底物 6-P-F-Asp 的积累，活细胞中的命中将进一步表征其 IC50、IC90、Ki 和计算特异性。化学将用于更好地了解 FraB 抑制剂的化学特征，并促进此外，定量结构-活性关系（QSAR）研究，以获得效力的结构基础。的命中，我们将使用 X 射线晶体学来确定 FraB 的原子分辨率结构，无论有无选择抑制剂预计成功完成这些目标将有助于命中识别和表征、先导化合物优化和发展至急需的窄谱的关键先决条件窄谱抗生素治疗非伤寒沙门氏菌病有两个主要优点：（i）限制。由正常微生物群破坏引起的副作用，以及 (ii) 避免选择抗菌药物我们设想未来可能会出现一种特定物种药物的混合物。用于治疗人类腹泻而不破坏健康微生物群的药物。该提案中确定的命中结果将成为该鸡尾酒的一个组成部分。