Assay Development for Inhibitors of the Essential sRNA-Sigma E Virulence Factors

必需 sRNA-Sigma E 毒力因子抑制剂的检测方法开发

• 批准号: 8051379
• 负责人: SARAH E ADES
• 金额: $ 14.8万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8051379
• 关键词: Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Physiology; Binding; Biological Assay; Burkholderia pseudomallei; Computer Systems Development; Cyclic Peptides; Data; Defect; Development; Escherichia coli; Future; Gene Mutation; Goals; Gram-Negative Bacteria; Growth; Health; Human; Immune response; Immune system; Institutes; Knowledge; Laboratories; Lead; Life; Membrane Proteins; Messenger RNA; Modification; Molecular; Molecular Bank; Molecular Target; Mutation; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Physiological; Production; Proteins; PubChem; Reagent; Research; Resistance; Role; Salmonella typhi; Screening procedure; Sigma Factor; Stress; Suppressor Mutations; System; Testing; Transcriptional Regulation; Vibrio cholerae; Virulence; Virulence Factors; Yersinia enterocolitica; assay development; base; cell envelope; env Gene Products; high throughput screening; inhibitor/antagonist; innovation; pathogen; porin; prevent; protein expression; prototype; reagent testing; research study; response; small molecule; transcription factor

DESCRIPTION (provided by applicant): Gram-negative pathogens can rapidly alter the composition of proteins in their envelope to adapt to a wide range of stresses, including challenges by the immune system and antibiotic treatment. A lack of knowledge of the molecular mechanisms by which these bacteria regulate the expression of envelope proteins limits understanding of interactions between pathogen and host, and prevents targeting crucial systems for development of new drugs. Our overall strategy is to identify inhibitors of molecular interactions required for the sE cell envelope sensing pathway, the major pathway used by Gram-negative bacteria to regulate outer membrane protein composition in response to challenges. This pathway is essential for viability in several important Gram-negative pathogens and required for virulence in others. The long-term goal of our research is to elucidate the role and mechanism of action of the sE pathway in bacterial physiology and pathogenesis. The objective of this proposal, which is the next required step in the attainment of our goal, is to develop an assay for high-throughput screening (HTS) to identify small molecule inhibitors of two key steps in the sE pathway: sE-dependent transcription, and regulation of porin mRNA levels by the protein Hfq in conjunction with sE-dependent sRNAs. Studies of genetic mutations in components of this pathway have led to a basic understanding of how the pathway functions. However, this approach is limited because it is difficult to distinguish direct functions of the pathway from the pleiotropic effects caused by the mutations. Small molecule inhibitors will enable experiments to test the function and molecular mechanism of this important pathway in greater detail. Guided by strong preliminary data, our objective will be attained by pursuing two specific aims: 1) optimize an assay for high-throughput screening to identify molecules that specifically inhibit two key components of the CE pathway; and 2) adapt and develop assays for secondary screening. Under the first aim, a primary assay that has been developed and tested in a pilot screen will be optimized for use in a high-throughput format. Under the second aim, existing assays will be adapted for use in secondary screens to eliminate false positive hits and to identify the molecules targeted by each inhibitor. The assays will be submitted to Molecular Libraries Production Centers Network (MLPCN) for implementation. Information about small molecule-target interactions obtained from these studies will be made available via PubChem. This proposal is innovative in its approach to simultaneously target a transcription factor, sE, and sRNA regulators that are required for a concerted physiological response to environmental challenges. All components of this pathway are highly conserved, so it is anticipated that inhibitors identified here can be used to study the role of this pathway in growth and virulence of many pathogens. These inhibitors will also provide lead compounds for future antibiotic development. PUBLIC HEALTH RELEVANCE: The proposed study will develop assays for high-throughput screening to identify inhibitors of a conserved bacterial pathway required to survive environmental and immune response challenges. Successful completion of these experiments and subsequent screening will provide a crucial set of reagents for testing how bacteria interact with their surroundings during growth and virulence. These reagents may also be lead compounds for development of new antibiotics.

描述（由申请人提供）：革兰氏阴性病原体可以快速改变其包膜中的蛋白质组成，以适应各种应激，包括免疫系统和抗生素治疗的挑战。由于缺乏对这些细菌调节包膜蛋白表达的分子机制的了解，限制了对病原体和宿主之间相互作用的理解，并阻碍了新药开发的靶向关键系统。我们的总体策略是识别 sE 细胞包膜传感通路所需的分子相互作用的抑制剂，这是革兰氏阴性细菌用来调节外膜蛋白组成以应对挑战的主要通路。该途径对于几种重要的革兰氏阴性病原体的生存能力至关重要，并且对于其他病原体的毒力也是必需的。我们研究的长期目标是阐明sE途径在细菌生理学和发病机制中的作用和作用机制。本提案的目标是开发一种高通量筛选 (HTS) 检测方法，以鉴定 sE 途径中两个关键步骤的小分子抑制剂：sE 依赖性转录，以及蛋白 Hfq 与 sE 依赖性 sRNA 结合对孔蛋白 mRNA 水平的调节，这是实现我们目标的下一个必要步骤。对该途径成分的基因突变的研究使人们对该途径如何发挥作用有了基本的了解。然而，这种方法是有限的，因为很难区分该途径的直接功能和突变引起的多效性效应。小分子抑制剂将使实验能够更详细地测试这一重要途径的功能和分子机制。在强有力的初步数据的指导下，我们的目标将通过追求两个具体目标来实现：1）优化高通量筛选测定法，以识别特异性抑制 CE 途径两个关键成分的分子； 2) 调整和开发二次筛选的检测方法。第一个目标是，在中试筛选中开发和测试的主要测定方法将被优化以用于高通量格式。根据第二个目标，现有的检测方法将适用于二次筛选，以消除假阳性命中并识别每种抑制剂靶向的分子。这些检测将提交给分子图书馆生产中心网络 (MLPCN) 进行实施。从这些研究中获得的小分子与靶标相互作用的信息将通过 PubChem 提供。该提案的创新之处在于它同时针对转录因子、sE 和 sRNA 调节因子，这些调节因子是对环境挑战做出协调一致的生理反应所必需的。该途径的所有组成部分都是高度保守的，因此预计此处鉴定的抑制剂可用于研究该途径在许多病原体的生长和毒力中的作用。这些抑制剂还将为未来抗生素的开发提供先导化合物。 公共健康相关性：拟议的研究将开发高通量筛选分析方法，以确定在环境和免疫反应挑战中生存所需的保守细菌途径的抑制剂。成功完成这些实验和随后的筛选将为测试细菌在生长和毒力过程中如何与其周围环境相互作用提供一套关键的试剂。这些试剂也可能是开发新抗生素的先导化合物。