Discovery and Development of Biofilm Inhibitors that Target c-di-GMP signaling

描述（由申请人提供）：据估计，超过75%的人类细菌感染与生物膜相关，但对控制生物膜形成的生化机制知之甚少。因此，对生物膜生理学的更深入理解的发展对基础细菌学和生物医学应用都具有深远的意义。特别是，生物膜形成的小分子抑制剂的发现提供了机会， 创建一套目标独立的工具，用于描绘控制生物膜形成的复杂调控级联，并开发生物膜相关感染的新疗法。我们最近开发了一种基于图像的高内容筛选方法，用于在高通量条件下可视化生物膜抑制剂。将该筛选技术应用于国家癌症研究所筛选文库表明，该工具可用于有效地从小分子筛选文库中鉴定生物膜抑制剂。在该资助范围内，我们的目标是进一步完善和优化该筛选平台，以包括人类病原体铜绿假单胞菌，并确定那些选择性影响cyclic-di-GMP信号通路的化合物;生物膜形成的主要调控机制。其次，我们的目标是探索这些探针的行动模式，通过一套分析，包括共聚焦显微镜，功能基因组学和分子遗传学。我们的进一步目标是检查生物膜抑制剂与传统抗生素疗法联合给药对细菌活力的影响，以探索它们作为治疗细菌生物膜感染的联合疗法的实用性。最后，我们将通过发酵优化研究开发可持续的先导化合物生产方法，为临床前开发准备先导候选项目。 公共卫生相关性：细菌感染仍然是发达国家住院的主要原因之一。在许多情况下，由于细菌生物膜的存在，这些感染更难以治疗。生物膜是一层层细菌“粘液”， 其覆盖生物表面（例如肺的内层）和人造表面（例如支架和导管）。生物膜产生了一种密集的蛋白质和糖基质，并保护细菌免受抗生素的作用。该项目旨在寻找小分子， 生物膜的形成，并使用这些化合物，以更好地了解生物膜形成的机制，使用最先进的荧光成像显微镜和小分子化学库。

DESCRIPTION (provided by applicant): It is estimated that over 75% of human bacterial infections are biofilm-related, yet relatively little is known about the biochemical mechanisms that govern biofilm formation. The development of a greater understanding of biofilm physiology therefore has profound implications for both basic bacteriology, and biomedical applications. In particular, the discovery of small molecule inhibitors of biofilm formation provides an opportunity to create a set of target-independent tools for delineating the complex regulatory cascade that control biofilm formation, and to develop new therapeutics for biofilm-related infections. We have recently developed an image-based, high content screening approach for visualizing biofilm inhibitors under high-throughput conditions. Application of this screening technology to the National Cancer Institute screening libraries has shown that this tool can be used to effectively identify biofilm inhibitors form small molecule screening libraries. Within the scope o this grant, we aim to further refine and optimize this screening platform to include the human pathogen Pseudomonas aeruginosa, and to identify those compounds that selectively impact cyclic-di-GMP signaling pathways; a major regulatory mechanism in biofilm formation. Secondly, we aim to explore the modes of action of these probes through a suite of analyses, including confocal microscopy, functional genomics and molecular genetics. We further aim to examine the effect on bacterial viability of co-dosing biofilm inhibitors with conventional antibiotic therapies to explore their utility as combination therapies for treatment of bacterial biofilm infections. Finally, we will develop sustainable methods of production of lead compounds through fermentation optimization studies to prepare lead candidate projects for pre-clinical development. PUBLIC HEALTH RELEVANCE: Bacterial infections remain one of the leading causes of hospitalization in the developed world. In many instances, these infections are made more difficult to treat by the presence of bacterial biofilms. Biofilms are layers of bacterial 'slime', which coat both biological surfaces (such as the lining of the lung) and artificial surfaces (such as stents and catheters). Biofilms create a dense matrix of proteins and sugars, and protect the bacteria from the action of antibiotics. This project aims to find small molecules that disrupt the formation of biofilms, and to use these compounds to better understand the mechanisms by which biofilms are formed, using state-of-the-art fluorescence imaging microscopy and small molecule chemical libraries.