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

描述(申请人提供)：革兰氏阴性病原体可以迅速改变其包膜蛋白的组成，以适应广泛的压力，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控包膜蛋白表达的分子机制的了解，限制了对病原体与宿主之间相互作用的了解，并阻碍了靶向新药开发的关键系统。我们的总体战略是寻找Se细胞膜感知途径所需的分子相互作用的抑制剂，Se细胞膜传感途径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途径。这一途径对几种重要的革兰氏阴性病原体的生存至关重要，对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途径在细菌生理和发病机制中的作用和机制。这项建议的目标是开发一种高通量筛选(HTS)的方法，以确定Se途径中两个关键步骤的小分子抑制物：依赖Se的转录，以及由蛋白质Hfq结合依赖Se的sRNAs调节porin mRNA水平。对该途径各组成部分的基因突变的研究使人们对该途径的功能有了基本的了解。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由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。在强大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)优化高通量筛选的检测方法，以确定特定抑制CE途径的两个关键成分的分子；2)调整和发展二次筛选的检测方法。在第一个目标下，已经开发并在中试屏幕上测试的初级分析将被优化，以用于高通量格式。在第二个目标下，现有的检测方法将被改装用于二次筛查，以消除假阳性，并识别每个抑制剂的靶向分子。检测结果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有关小分子-目标相互作用的信息将通过PubChem提供。这一建议的创新之处在于，它同时针对转录因子、Se和sRNA调控因子，这是对环境挑战做出协调生理反应所必需的。该途径的所有组成部分都是高度保守的，因此可以预期，本研究中发现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的抗生素开发提供先导化合物。 与公共健康相关：拟议的研究将开发高通量筛选的分析方法，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物。成功完成这些实验和随后的筛选将提供一套关键的试剂，用于测试细菌在生长和毒力过程中如何与环境相互作用。这些试剂也可能是开发新抗生素的先导化合物。

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.