Innate immunity-based screen for bacterial type III secretion system inhibitors

基于先天免疫的细菌 III 型分泌系统抑制剂筛选

• 批准号: 9044408
• 负责人: Victoria Auerbuch Stone
• 金额: $ 7.03万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9044408
• 关键词: Actins; Antibiotics; Bacterial Typing; Biochemical; Biological Assay; Biological Factors; Cell Communication; Cell Line; Cell physiology; Cells; Cellular Morphology; Cessation of life; Clinic; Data; Detection; Development; Disease; Dyes; Effector Cell; Family; Fluorescence Resonance Energy Transfer; Foundations; Future; Future Generations; Goals; Gram-Negative Bacteria; Health; Housing; Human; Immune system; Immunofluorescence Microscopy; In Vitro; Infection; Lactamase; Lead; Left; Libraries; Luciferases; Mammalian Cell; Marketing; Membrane; Methods; Microbe; Microscopy; Modeling; Modern Medicine; Molecular Bank; Molecular Target; Natural Immunity; Organism; Pasteurella pseudotuberculosis; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Production; Proteins; Reporter; Reproducibility; Research; Resistance; Resistance development; Salmonella; Shigella; Staging; Therapeutic; Transfection; Type III Secretion System Pathway; United States National Institutes of Health; Validation; Virulence; Work; Yersinia; Yersinia enterocolitica; antimicrobial; antimicrobial drug; appendage; bacterial resistance; base; biochemical tools; design; drug candidate; enteropathogenic Escherichia coli; high throughput screening; improved; in vivo; inhibitor/antagonist; marine natural product; member; next generation; novel; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; prevent; promoter; response; scaffold; screening; small molecule; stable cell line; tool; transcription factor

DESCRIPTION (provided by applicant): The type III secretion system (T3SS) is a bacterial appendage required by dozens of pathogens to cause disease, including Salmonella, EPEC, Shigella, and Yersinia. Collectively, these pathogens cause over 200 million cases of human illness and well over half a million deaths per year. Yet these and other bacterial pathogens are developing resistance to currently available antibiotics at an alarming rate. As only two new classes of antibiotics have reached the market since 1962, new therapeutics are critically needed to support modern medicine for future generations. The T3SS is well-conserved across a large number of bacterial species yet is specifically expressed by pathogens. This makes the T3SS an optimal target for antimicrobial agents that can hinder the pathogenesis of T3SS-expressing pathogens without causing large disruptions to the microbiota. While T3SS inhibitors have been identified, few have known molecular targets and none have reached the clinic. The function of the T3SS is to inject bacterial effector proteins inside target host cells. Once inside host cells, these effector proteins manipulate normal host cell processes, to the benefit of the pathogen. We have devised a novel strategy for identifying small molecule inhibitors that can disrupt the ability of the T3SS to functionally interact with host cells. This assay relies on the ability of mammalian cells to activate the host transcription factor NFκB in response to a functional T3SS, letting host cells serve as an indicator of whether a small molecule can inhibit T3SS function. In Aim 1, we will exploit this NFκB activation assay to develop a high throughput screen (HTS) to identify T3SS inhibitors, using Yersinia pseudotuberculosis as a model T3SS-expressing pathogen. To achieve this, we will generate a stably-transfected NFκB luciferase reporter cell line that can support a robust and reproducible HTS. In addition, we will develop a rapid secondary screen to eliminate compounds that inhibit the NFκB pathway, allowing us to focus our efforts on compounds that specifically target the T3SS. In Aim 2, we will develop and vet an experimental pipeline that (i) will serve to validate the T3SS inhibitory activity of lead compounds and (ii) will allow rapid identification of the stage of type III secretion targeted by lead compounds,-i.e., T3SS assembly, T3SS pore formation on host membranes, T3SS translocation of effector proteins inside host cells, and activity of T3SS effector proteins inside host cells. Upon completion of this work, we will carry out the HTS screen and experimental pipeline described in this proposal, using our in-house natural products and synthetic compound libraries (over 55,000 compounds). In addition, we will submit our screen for Fast Track entry into the NIH Roadmap Molecular Libraries Probe Production Centers Network. This will enable identification of a suite of T3SS inhibitors that target different stages of type III secretion, whch will serve as the foundation for future development of a biochemical toolbox of T3SS inhibitors with known molecular targets that will be used as research probes and scaffolds for novel therapeutics.

描述(申请人提供)：III型分泌系统(T3SS)是一种细菌附属物，包括沙门氏菌、EPEC、志贺氏菌和耶尔森氏菌等数十种病原体致病所必需的。总体而言，这些病原体每年导致2亿多例人类疾病和50多万人死亡。然而，这些和其他细菌病原体正在以惊人的速度对目前可用的抗生素产生抗药性。自1962年以来，只有两类新的抗生素上市，因此迫切需要新的疗法来支持未来几代人的现代医学。T3SS在许多细菌物种中都被很好地保守，但却被病原体特异性地表达。这使得T3SS成为抗菌剂的最佳靶点，这些抗菌剂可以在不对微生物区系造成大破坏的情况下阻止T3SS表达病原体的发病。虽然已经确定了T3SS抑制剂，但很少有已知的分子靶点，也没有人进入临床。T3SS的功能是将细菌效应蛋白注入目标宿主细胞内。一旦进入 在宿主细胞中，这些效应蛋白操纵正常的宿主细胞过程，对病原体有利。我们设计了一种新的策略来识别小分子抑制剂，这些小分子抑制剂可以破坏T3SS与宿主细胞功能相互作用的能力。这一检测依赖于哺乳动物细胞激活宿主转录因子NFκB以响应功能性T3SS的能力，让宿主细胞作为小分子是否可以抑制T3SS功能的指示器。在目标1中，我们将以假结核耶尔森菌作为表达T3SS的模型病原菌，利用这种核因子κB激活试验建立高通量筛选方法来鉴定T3SS抑制剂。为了实现这一点，我们将建立一个稳定转染的NFκB荧光素酶报告细胞系，该细胞系能够支持健壮和可重复的高温超导。此外，我们将开发一种快速二次筛选来消除抑制NFκB途径的化合物，使我们能够将精力集中在专门针对T3SS的化合物上。在目标2中，我们将开发和审查一条实验管道，该管道(I)将用于验证先导化合物的T3SS抑制活性，(Ii)将允许快速识别先导化合物针对的III型分泌阶段，即T3SS组装、T3SS在宿主膜上的孔形成、T3SS效应蛋白在宿主细胞内的移位以及T3SS效应蛋白在宿主细胞内的活性 宿主细胞。在这项工作完成后，我们将使用我们内部的天然产品和合成化合物库(超过55,000种化合物)，进行本提案中描述的高温超导筛选和实验管道。此外，我们将提交我们的快速通道进入NIH路线图分子图书馆探测器生产中心网络的屏幕。这将使我们能够识别一套针对III型分泌不同阶段的T3SS抑制剂，这将成为未来开发具有已知分子靶点的T3SS抑制剂生化工具箱的基础，该工具箱将用作新疗法的研究探针和支架。