High Throughput Screening of Inhibitors of Pyoverdine Production

说明(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特别有问题，是医院感染的常见原因。广为人知的抗药性细菌的流行表明，需要确定新的目标和确定新的抑制策略，这些策略可以开发成新的抗生素疗法。铜绿假单胞菌使用非核糖体多肽合成簇来产生铁摄取所必需的多肽铁载体。寄主生物体中的铁是一种限制性营养物质，大量的动植物研究表明，吡哆醇在毒力中发挥着重要作用。因此，负责合成吡喃佛定的14种蛋白质中的任何一种都是有效的抑制目标，以防止感染的建立。我们已经开发了一种用于PvdQ的生化分析方法，PvdQ是一种周质酰基酶，负责吡喃呋喃的化学成熟的后期步骤。二次和三次试验将验证初步筛选试验的结果，并在细胞生长试验中评估化合物的生存能力。小型化学库的初步筛选显示了非常好的信噪比和重复性统计数据，分子库和探针生产中心网络的筛选可能会识别阻止PvdQ的新化合物。在这项工作中确定的化合物将被用来进一步了解PvdQ和相关蛋白在假单胞菌发病中的作用，长期目标是优化这些化合物作为吡喃佛丁生产的特定抑制剂。 公共卫生相关性：铜绿假单胞菌是一种人类病原体，需要生物化学系统才能获得铁以确定感染。已经开发了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。

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.