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（由申请人提供）：III型分泌系统（T3SS）是数十种病原体引起疾病所需的细菌附属物，包括沙门氏菌，EPEC，志贺氏菌和耶尔森氏菌。这些病原体每年总共造成2亿多例人类疾病和50多万例死亡。然而，这些和其他细菌病原体正以惊人的速度对现有抗生素产生耐药性。由于自1962年以来只有两种新型抗生素进入市场，因此迫切需要新的治疗方法来支持后代的现代医学。T3SS在大量细菌物种中保守性良好，但在病原体中特异性表达。这使得T3SS成为抗菌药物的最佳靶点，可以阻止表达T3SS的病原体的发病机制，而不会对微生物群造成大的破坏。虽然已经确定了T3SS抑制剂，但很少有已知的分子靶点，而且没有一个达到临床。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