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节的理解，但尚未广泛追求针对细菌发病机理必不可少的调节系统的抗生素的发展。我们假设抑制保守的pHOP毒力调节的小分子可能构成有效的抗生素。 pHOP调节是细胞内存活和多种病原体毒力所需的基因的重要调节因子，并且在沙门氏菌血清鼠伤寒沙门氏菌的模型生物中的表征最佳。在这里，我们建议开发高吞吐量分子筛选（HTS）测定，以鉴定PHOP调节的化学抑制剂。特别是，我们的策略将旨在筛选小分子，这些分子同时抑制来自PHOP激活启动子的报告基因的表达，同时增加了pHOP抑制启动子的基因表达。在特定的目标1中，我们将开发一系列重组pHOP激活和pHOP抑制的启动子重型 - 重孢子，并量化这些记者在血清中生长在pHOP诱导和非诱导条件下生长的血清鼠伤寒中的表达。在特定的目标2中，我们将通过选择具有最高信噪比比率的单个PHOP激活和单个PHOP抑制启动子来配置HTS的测定法，并在96和384井格式中驾驶测定法。在此提案中，我们还概述了在初始HTS屏幕中确定的二级评估和优先级的详细顺序策略。多药物抗性细菌是全球发病率和死亡率的重要原因，并且具有克服我们当前的生物形式的能力，所有这些都需要发展新型抗生素类别。拟议的测定方法可以鉴定具有独特抗生素特性的小分子，以及针对细胞内病原体的新型作用机理，包括伤寒的药物抗药性原因，这是全球发病率和死亡率的主要原因，以及瘟疫，耶尔西尼亚pestis，耶尔森尼亚pestis，耶稣基因pestis，Yersinia pestis，Yersinia pestis，Yersinia pestis，Yersinia pestis，pesterrorist的巨大威胁。在未来的研究中，使用拟议测定法鉴定的化合物可能会发展为一种用于治疗许多细菌感染的新药物，包括几种A和B类优先病原体。