Targeting Bacterial Virulence Transcription Factors, a Novel Antibiotic Approach

针对细菌毒力转录因子，一种新的抗生素方法

• 批准号: 7893519
• 负责人: George A Garcia
• 金额: $ 24.33万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893519
• 关键词: Acute; Address; Antibiotics; Bacteria; Biological Assay; Biological Factors; Biological Models; Categories; Cells; Chemicals; Child; Developed Countries; Development; Diarrhea; Disease; Drug resistance; Evaluation; Family; Food; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Growth; Human; Immune system; In Vitro; Individual; Knock-out; Lead; Libraries; Life; Malnutrition; Michigan; Multi-Drug Resistance; National Institute of Allergy and Infectious Disease; Organism; Pathogenesis; Pharmaceutical Preparations; Predisposition; Property; Published Comment; Regulation; Resistance development; Shigella; Shigella Infections; Shigella flexneri; Transcription Coactivator; Transcription factor genes; Universities; Validation; Virulence; Virulence Factors; Work; analog; assay development; base; design; high throughput screening; in vitro Assay; mental development; novel; pathogen; pathogenic bacteria; pressure; public health relevance; resistant strain; transcription factor; waterborne

DESCRIPTION (provided by applicant): Many pathogenic bacteria express virulence genes that are required for infectivity and pathogenesis. Virulence factors are attractive targets for antibiotic discovery as there should be less selective pressure for the organisms to develop resistance since virulence factors are not essential for bacterial growth. However, targeting a large number of individual virulence factors makes this approach difficult. Expression of these genes is often controlled by specific virulence gene transcription factors of the AraC family. Inhibition of these transcription factors could represent a novel and general approach to antibiotic discovery. While much is known about the control of transcription in bacteria, those transcription factors involved in virulence are poorly understood. One example is Shigella flexneri, in which VirF, an AraC-type transcriptional regulator, is responsible for the expression of all downstream virulence factors that control intracellular invasion and cell-to- cell spread. Shigella infects ca. 165 million people and claims more than one million lives per year worldwide. While shigellosis has been considered to be a disease of the "third world", like many other highly contagious diseases it is rapidly spreading to developed countries. The emergence of drug and multi-drug resistant strains of Shigella emphasize the need for novel antibiotic development. Gene knock-out studies have validated that inhibition of VirF expression is sufficient to block the invasion cascade of Shigella in the host and thereby increase susceptibility to the host immune system. Our goals for this project are twofold. First, we wish to demonstrate the feasibility of targeting the regulation of virulence gene expression as a novel antibiotic approach using VirF as our model system. Second, the further development of validated hits against VirF will lead to novel agents against shigellosis, a desirable goal in itself as discussed above. The specific aims for this R21 application are two-fold. Aim 1: The development of assays for VirF, and adaptation of one or more assays for high throughput screening (HTS). HTS will be done on a chemically-diverse library of compounds (ca. 155,000 + 16,000 unique natural product extracts) held by the University of Michigan, Center for Chemical Genomics. Aim 2: "Hits" from HTS will be manually screened for potential "drugability" properties. Hits with appropriate properties will be further characterized via a number of in vitro assays. These assays are designed to validate the hits, provide guidance for subsequent lead optimization and also to probe mechanism(s) of inhibition. These studies will also help to elucidate the mechanism of action of VirF. In subsequent work, we will conduct a "hit-to-lead" campaign involving iterative cycles of analogue design, synthesis, and evaluation. We will also investigate the basic mechanisms of this poorly understood transcription factor. These studies will provide proof of principle for targeting virulence gene transcriptional activators with application to many human pathogens. This proposal has been revised to address the comments of the reviewers and to include progress that we have made on HTS assay development. PUBLIC HEALTH RELEVANCE: Relevance In the developing world, severe diarrhea is a killer, taking the lives of 2 to 3 million children every year. Persistent cases of diarrhea cause malnutrition, retard growth, and delay mental development. Shigella flexneri is one of the main causative agents of diarrheal disease. While shigellosis has been considered to be a disease of the "third world", like many other highly contagious diseases it is rapidly spreading to developed countries. Importantly, the emergence of a number of drug resistant strains of Shigella emphasizes the need for novel antibiotic development. This project will demonstrate the feasibility of targeting bacterial virulence- regulating transcription factors toward the discovery of novel antibiotics.

性状（由申请人提供）：许多病原菌表达感染性和致病性所需的毒力基因。毒力因子是抗生素发现的有吸引力的目标，因为由于毒力因子对于细菌生长不是必需的，因此生物体产生抗性的选择压力应该较小。然而，针对大量个体毒力因子使得这种方法变得困难。这些基因的表达通常由AraC家族的特异性毒力基因转录因子控制。这些转录因子的抑制可能代表了抗生素发现的一种新的和通用的方法。虽然对细菌中的转录控制了解很多，但对涉及毒力的那些转录因子知之甚少。一个例子是福氏志贺菌，其中VirF，一种AraC型转录调节因子，负责控制细胞内侵入和细胞间传播的所有下游毒力因子的表达。志贺氏菌感染ca. 1.65亿人，每年在全世界夺走100多万人的生命。虽然志贺氏菌病一直被认为是“第三世界”的疾病，但与许多其他高度传染性疾病一样，它正在迅速蔓延到发达国家。志贺氏菌耐药菌株和多重耐药菌株的出现强调了开发新型抗生素的必要性。基因敲除研究已经证实，抑制VirF表达足以阻断志贺氏菌在宿主中的侵袭级联，从而增加对宿主免疫系统的易感性。我们对这个项目的目标是双重的。首先，我们希望以VirF为模型系统，证明靶向调节毒力基因表达作为一种新型抗生素方法的可行性。第二，进一步开发针对VirF的经验证的命中物将导致针对志贺氏菌病的新型药剂，这本身就是如上所述的理想目标。R21应用的具体目标有两个方面。目的1：开发VirF的检测方法，并将一种或多种检测方法用于高通量筛选（HTS）。HTS将在一个化学多样的化合物库上进行（约100种）。155，000 + 16，000独特的天然产品提取物）由密歇根大学化学基因组学中心持有。目的2：将手动筛选HTS的“命中”，以确定潜在的“可药性”特性。具有适当性质的命中物将通过许多体外测定进一步表征。这些试验旨在验证命中，为后续电极导线优化提供指导，并探索抑制机制。这些研究也将有助于阐明VirF的作用机制。在随后的工作中，我们将进行一个“命中领先”的运动，涉及类似物设计，合成和评估的迭代周期。我们还将研究这种知之甚少的转录因子的基本机制。这些研究将为靶向毒力基因转录激活因子应用于许多人类病原体提供原理证明。已对本提案进行了修订，以解决评审员的意见，并纳入我们在HTS检测开发方面取得的进展。 在发展中国家，严重腹泻是一个杀手，每年夺去200万至300万儿童的生命。持续的腹泻病例会导致营养不良、生长迟缓和智力发育迟缓。福氏志贺菌是引起腹泻的主要病原菌之一。虽然志贺氏菌病一直被认为是“第三世界”的疾病，但与许多其他高度传染性疾病一样，它正在迅速蔓延到发达国家。重要的是，志贺氏菌耐药菌株的出现强调了开发新型抗生素的必要性。这个项目将证明以细菌毒力调节转录因子为靶点发现新型抗生素的可行性。