High Throughput Screening of Inhibitors of Pyoverdine Production

吡维定生产抑制剂的高通量筛选

• 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
• 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

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尤其有问题，并且是医院感染的常见原因。众所周知的耐药细菌的流行表明需要表征新的靶点，并确定可能开发成新的抗生素疗法的新的抑制策略。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。铁在宿主生物体中是一种限制性营养素，许多动物和植物研究已经证明了绿脓菌荧光素在毒力中的重要作用。因此，负责合成绿脓菌荧光素的14种蛋白质中的任何一种都是用于抑制以防止感染建立的可行靶标。我们已经开发了一种与PvdQ一起使用的生物化学测定法，PvdQ是负责绿脓菌荧光素化学成熟中的后期步骤的周质酰化酶。二级和三级试验将验证初级筛选试验的结果，并在细胞生长试验中评估化合物的活力。用小的化学文库进行初步筛选显示出非常好的信噪比和再现性统计，并且在分子文库和探针生产中心网络进行筛选可能会鉴定出阻断PvdQ的新型化合物。在这项工作中鉴定的化合物将用于进一步理解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绿脓菌荧光素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，需要生化系统来获得铁以建立感染。已经开发了一种测定法来鉴定能够阻断该过程的化学探针。高通量筛选将促进这些抑制性化合物的鉴定，作为开发新型抗生素的第一步。