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

描述(申请人提供)：据估计，超过75%的人类细菌感染与生物膜有关，但对生物膜形成的生化机制知之甚少。因此，更好地了解生物膜生理学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一个机会 创建一套目标无关的工具，用于描述控制生物膜形成的复杂调控级联反应，并开发治疗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、高含量的筛选方法，用于在高通量条件下可视化生物膜抑制剂。该筛选技术在国家癌症研究所筛选文库中的应用表明，该工具可以有效地从小分子筛选文库中识别生物膜抑制剂。在这笔赠款的范围内，我们的目标是进一步细化和优化这一筛选平台，以包括人类病原体铜绿假单胞菌，并识别那些选择性地影响环-二-GMP信号通路的化合物，这是生物被膜形成的主要调控机制。其次，我们的目标是通过一系列分析来探索这些探针的作用模式，包括共聚焦显微镜、功能基因组学和分子遗传学。我们进一步的目标是检测联合使用生物膜抑制剂和传统抗生素疗法对细菌生存的影响，以探索它们作为联合疗法治疗细菌生物膜感染的有效性。最后，我们将通过发酵优化研究开发可持续生产先导化合物的方法，为临床前开发准备领先的候选项目。 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，细菌生物膜的存在使这些感染变得更加难以治疗。生物膜是细菌的一层“黏液”， 它覆盖了生物表面(如肺的内层)和人造表面(如支架和导管)。生物膜创造了一种由蛋白质和糖组成的致密基质，并保护细菌免受抗生素的影响。这个项目的目标是找到能够扰乱 利用最先进的荧光成像显微镜和小分子化学库，研究生物膜的形成，并利用这些化合物更好地了解生物膜形成的机制。

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.