High Throughput Screening of Inhibitors of Pyoverdine Production

吡维定生产抑制剂的高通量筛选

基本信息

批准号：
8109333
负责人：
ANDREW M GULICK
金额：
$ 4.75万
依托单位：
HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8109333
关键词：
4-nitrophenol Acyl Coenzyme A Affinity Amides Amino Acids Anabolism Animals Antibiotic Therapy Antibiotics Bacteria Binding Biochemical Biological Assay Carbon Dioxide Cells Chemicals Chemistry Chromogenic Substrates Coenzyme A Ligases Complex Coupled Defect Development Enzymes Esters Evolution Excision Fatty Acids Genes Goals Growth Human Immunocompromised Host Infection Iron Lead Libraries Link Modification Molecular Bank Molecular Probes Molecular Weight Monitor Myristates Noise Nosocomial Infections Nucleic Acids Nutrient Organism Pathogenesis Pathway interactions Peptides Pharmaceutical Preparations Plants Play Prevalence Process Production Proteins Pseudomonas Pseudomonas aeruginosa Reaction Reproducibility Role Screening procedure Serum Siderophores Signal Transduction Solubility Structure System United States National Institutes of Health Virulence Work amidase base cell growth deacylation drug resistant bacteria high throughput screening inhibitor/antagonist mutant novel pathogen pathogenic bacteria peptide synthase periplasm prevent public health relevance pyoverdin siderophore receptors small molecule libraries statistics synthetic peptide tool uptake

项目摘要

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa, a gram-negative bacterial species, is a human pathogen that is particularly problematic for immunocompromised patients and is a common cause of nosocomial infections. The well-publicized prevalence of drug resistant bacteria demonstrates a need for the characterization of new targets and for the identification of novel inhibition strategies that may be developed into new antibiotic therapies. Pseudomonas aeruginosa uses a non-ribosomal peptide synthetic cluster to produce a peptide siderophore that is necessary for iron uptake. Iron in the host organism is a limiting nutrient and numerous animal and plant studies have demonstrated the important role that pyoverdine plays in virulence. Thus, any of the 14 proteins responsible for the synthesis of pyoverdine are viable targets for inhibition to prevent the establishment of an infection. We have developed a biochemical assay for use with PvdQ, the periplasmic acylase that is responsible for a late step in the chemical maturation of pyoverdine. Secondary and tertiary assays will validate that results from the primary screening assay and assess the viability of compounds in a cell growth assay. Preliminary screening with a small chemical library demonstrates very good signal to noise and reproducibility statistics and screening at the Molecular Libraries and Probe Production Centers Network will likely identify novel compounds that block PvdQ. Compounds identified in this effort will be used to further our understanding of the role of PvdQ and related proteins in Pseudomonas pathogenesis with a long term goal of optimizing these compounds as specific inhibitors of pyoverdine production. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a human pathogen that requires biochemical systems for the acquisition of iron to establish an infection. An assay has been developed to identify chemical probes that are able to block this process. High-throughput screening will facilitate the identification of these inhibitory compounds as an initial step towards the development of novel antibiotics.

描述（由申请人提供）：铜绿假单胞菌是一种革兰氏阴性细菌，是一种人类病原体，对免疫功能低下的患者来说尤其成问题，并且是医院感染的常见原因。广为人知的耐药细菌的流行表明需要表征新靶点并确定可开发成新抗生素疗法的新抑制策略。铜绿假单胞菌使用非核糖体肽合成簇来产生铁吸收所必需的肽铁载体。宿主生物体中的铁是一种限制性营养物质，大量的动物和植物研究已经证明了pyoverdine在毒力中发挥的重要作用。因此，负责合成吡弗定的 14 种蛋白质中的任何一种都是抑制感染的可行靶标。我们开发了一种与 PvdQ 一起使用的生化测定法，PvdQ 是一种周质酰基转移酶，负责吡维丁化学成熟的后期步骤。二级和三级测定将验证初级筛选测定的结果，并评估化合物在细胞生长测定中的活力。使用小型化学库进行的初步筛选显示出非常好的信噪比和重现性统计数据，并且在分子库和探针生产中心网络进行的筛选可能会识别出阻断 PvdQ 的新型化合物。这项工作中鉴定出的化合物将用于进一步了解 PvdQ 和相关蛋白在假单胞菌发病机制中的作用，长期目标是优化这些化合物作为吡维定生产的特异性抑制剂。公共卫生相关性：铜绿假单胞菌是一种人类病原体，需要生化系统来获取铁来建立感染。已经开发出一种检测方法来识别能够阻止这一过程的化学探针。高通量筛选将有助于鉴定这些抑制化合物，作为开发新型抗生素的第一步。