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

沙门氏菌 PhoP 调节子抑制剂小分子筛选的开发

• 批准号: 7678707
• 负责人: JASON B HARRIS
• 金额: $ 4.4万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-05 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678707
• 关键词: Animal Model; Antibiotics; Award; Bacterial Infections; Biological; Biological Assay; Biological Models; Categories; Chemicals; Class; Computer Systems Development; Condition; Development; Drug Delivery Systems; Drug resistance; Evaluation; Funding; Future; Gene Expression; Gene Expression Regulation; Genes; Image; Magnesium; Molecular; Molecular Bank; Morbidity - disease rate; Multi-Drug Resistance; Numbers; Pathogenesis; Plague; Principal Investigator; Property; Recombinants; Regulation; Regulator Genes; Regulon; Reporter; Reporter Genes; Research Institute; Salmonella; Salmonella enterica; Screening procedure; Series; Signal Transduction; Site; System; Time; Typhoid Fever; United States National Institutes of Health; Virulence; Yersinia pestis; base; biodefense; design; drug resistant bacteria; high throughput screening; high throughput technology; inhibitor/antagonist; microbial; mortality; novel; pathogen; programs; promoter; small molecule; tool

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.

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追求。我们推测，抑制保守的PhoP毒力调节子的小分子可能构成有效的抗生素。PhoP调节子是许多病原体的细胞内存活和毒力所需的基因的重要调节子，并且在沙门氏菌鼠伤寒血清型的模式生物中得到了最好的表征。在这里，我们建议开发一个高通量的分子筛选（HTS）检测，以确定化学抑制剂的PhoP调节子。特别是，我们的策略将被设计为筛选小分子，同时抑制报告基因从PhoP激活的启动子的表达，同时增加基因从PhoP抑制的启动子的表达。在具体目标1中，我们将开发一系列的重组PhoP激活和PhoP抑制的启动子-报告融合，并量化这些报告在PhoP诱导和非诱导条件下生长的鼠伤寒血清型中的表达。在特定目标2中，我们将通过选择具有最高信号背景比的单个PhoP激活和单个PhoP抑制的启动子并在96和384孔格式中试点试验来配置HTS测定。在本提案中，我们还概述了一个详细的顺序策略，用于对初始HTS筛选中确定的活性化合物进行二次评价和优先排序。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。所提出的测定法可以鉴定具有独特抗生素性质和针对细胞内病原体的新作用机制的小分子，所述细胞内病原体包括伤寒的耐药性原因（其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（其是重大的生物恐怖主义威胁）。在未来的研究中，使用所提出的测定法鉴定的化合物可能被开发成一类新的药物，用于治疗许多细菌感染，包括几种A类和B类优先病原体。