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

基本信息

批准号：
8696691
负责人：
Victoria Auerbuch Stone
金额：
$ 37.29万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
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/8696691
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 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 public health relevance response scaffold screening small molecule stable cell line tool transcription factor

项目摘要

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 NF?B 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 NF?B 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 NF?B 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 NF? B 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（由申请人提供）：III型分泌系统（T3SS）是数十种病原体所要求的细菌附属物，以引起疾病，包括沙门氏菌，EPEC，Shigella和Yersinia。总的来说，这些病原体会导致超过2亿例人类疾病，每年造成超过半百万的死亡。然而，这些和其他细菌病原体正在以惊人的速度发展对当前可用的抗生素的抗性。自1962年以来，由于只有两种新的抗生素已经进入了市场，因此需要新的治疗方法来支持后代的现代医学。 T3S在许多细菌种类中保存良好，但由病原体特别表达。这使T3SS成为抗菌剂的最佳靶标，可以阻止表达T3SS的病原体的发病机理，而不会引起对微生物群的大破坏。尽管已经鉴定出T3SS抑制剂，但很少有已知的分子靶标，也没有达到诊所。 T3SS的功能是在靶宿主细胞中注入细菌效应蛋白。室内宿主细胞，这些效应蛋白操纵正常的宿主细胞过程，以使病原体受益。我们已经设计了一种新的策略来鉴定可能破坏T3SS与宿主细胞相互作用的能力的小分子抑制剂。该分析依赖于哺乳动物细胞对功能性T3SS激活宿主转录因子NF？b的能力，这使宿主细胞用作指示小分子是否可以抑制T3SS功能。在AIM 1中，我们将利用该NF？B激活测定法开发高吞吐量筛选（HTS），以使用耶尔森氏菌为抑制T3SS的病原体，以识别T3SS抑制剂。为了实现这一目标，我们将生成稳定的转染的NF？B荧光素酶报告基细胞系，该细胞系可以支持可靠且可重现的HTS。此外，我们将开发一个快速的次级筛选，以消除抑制NF的化合物？ B途径，使我们能够将精力集中在专门针对T3SS的化合物上。在AIM 2中，我们将开发和审查（i）将有助于验证铅化合物的T3SS抑制活性和（ii）将允许铅化合物靶向的III型分泌的阶段，-i.e。，T3SS组装，T3Ss组装，T3SS孔对宿主膜的protistor and Trance and trancors of t3s的效应，T3SS组装的T3SS组装对T3S的活性，T3SS组装的T3S protecors and t3s transe and trancors and t3s transe效应的T3S型号的效果，里面的蛋白质宿主细胞。完成这项工作后，我们将使用我们的内部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HTS屏幕和实验管道。此外，我们将提交屏幕，以便快速进入NIH路线图分子库探测生产中心网络。这将使能够鉴定针对III型分泌不同阶段的T3SS抑制剂套件，WHCH将成为未来开发具有已知分子靶标的T3SS抑制剂的生化工具箱的基础，这些工具具有已知的分子靶标，这些靶标将用作新疗法的研究探针和支架。