Development of a small molecule screen for PhoP regulon inhibitors in Salmonella

描述（由申请人提供）：尽管高通量技术增强了我们对细菌毒力基因调控的理解，但针对细菌发病机制所必需的调控系统的抗生素的开发尚未得到广泛的追求。我们假设抑制保守的PhoP毒力调控子的小分子可能构成有效的抗生素。PhoP调控子是许多病原体细胞内存活和毒力所需基因的重要调控因子，在沙门氏菌血清型鼠伤寒沙门氏菌模式生物中得到了最好的表征。在这里，我们建议开发一种高通量分子筛选（HTS）方法来鉴定PhoP调控子的化学抑制剂。特别是，我们的策略将被设计用于筛选同时抑制phop激活启动子中报告基因表达，同时增加phop抑制启动子中基因表达的小分子。在Specific Aim 1中，我们将开发一系列重组phop激活和phop抑制的启动子-报告子融合体，并量化这些报告子在诱导和非诱导条件下生长的血清鼠伤寒菌中的表达。在具体目标2中，我们将通过选择具有最高信号背景比的单个phop激活启动子和单个phop抑制启动子并在96和384孔格式中进行试验来配置HTS检测。在本提案中，我们还概述了在初始HTS筛选中鉴定的活性化合物的二次评估和优先排序的详细顺序策略。多重耐药细菌是全球发病率和死亡率的重要原因，并有能力克服我们目前的生物防御，所有这些都需要开发新的抗生素类别。所提出的检测方法可以鉴定出具有独特抗生素特性的小分子和对抗细胞内病原体的新作用机制，包括伤寒的耐药性病原体，这是世界范围内发病率和死亡率的主要原因，以及鼠疫的病原体，鼠疫耶尔森氏菌，一个重大的生物恐怖威胁。在未来的研究中，利用所提出的检测方法鉴定的化合物可能会发展成为一类新的靶向药物，用于治疗许多细菌感染，包括几种a类和B类优先病原体。

DESCRIPTION (provided by applicant): Although high-throughput technologies have enhanced our understanding of bacterial virulence gene regulation, the development of antibiotics that target the regulatory systems that are essential for bacterial pathogenesis has not been extensively pursued. We hypothesize that small molecules inhibiting the conserved PhoP virulence regulon may constitute effective antibiotics. The PhoP regulon is an essential regulator of the genes required for intracellular survival and virulence of a number of pathogens and has been best characterized in the model organism of Salmonella enterica serovar Typhimurium. Here, we propose to develop a high throughput molecular screening (HTS) assay to identify chemical inhibitors of the PhoP regulon. In particular, our strategy will be designed to screen for small molecules that simultaneously inhibit the expression of reporter genes from PhoP-activated promoters, while increasing the expression of genes from PhoP-repressed promoters. In Specific Aim 1, we will develop a series of recombinant PhoP-activated and PhoP-repressed promoter-reporter fusions, and quantify the expression of these reporters in serovar Typhimurium grown in PhoP-inducing and non-inducing conditions. In Specific Aim 2, we will configure the assay for HTS by selecting a single PhoP-activated and a single PhoP-repressed promoter with the highest signal-to- background ratios and piloting the assay in 96 and 384 well formats. In this proposal we also outline a detailed sequential strategy for the secondary evaluation and prioritization of active compounds identified in the initial HTS screen. Multi-drug resistant bacteria are important causes of global morbidity and mortality and have the capacity to overcome our current biodefense, all of which necessitate the development of novel antibiotic classes. The proposed assay may identify small molecules with unique antibiotic properties and a novel mechanism of action against intracellular pathogens including the drug- resistant cause of typhoid fever, a major cause of morbidity and mortality worldwide, as well the causative agent of the plague, Yersinia pestis, a significant bioterrorist threat. In future studies, compounds identified using the proposed assays may be developed into a new class of drugs targeted for the treatment of many bacterial infections, including several category A and B priority pathogens.