High Throughput Screening of Inhibitors of Pyoverdine Production

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，是医院感染的常见原因。众所周知，耐药细菌的普遍存在表明，需要确定新的靶点，并确定可能发展成新的抗生素治疗的新的抑制策略。铜绿假单胞菌使用非核糖体肽合成簇来产生铁摄取所必需的肽铁载体。铁在宿主体内是一种限制性营养物质，许多动物和植物研究已经证明了吡啶在毒力中起着重要作用。因此，负责pyoverdine合成的14种蛋白质中的任何一种都是抑制以防止感染建立的可行目标。我们已经开发了一种用于PvdQ的生化试验，PvdQ是一种质周酰化酶，负责pyoverdine化学成熟的最后一步。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。用一个小的化学文库进行初步筛选，显示出非常好的信号噪声和可重复性统计，在分子文库和探针生产中心网络进行筛选，可能会发现阻断PvdQ的新化合物。在这项工作中鉴定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，并长期优化这些化合物作为pyoverdine生产的特异性抑制剂。

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.