Targeting Bacterial Virulence Transcription Factors, a Novel Antibiotic Approach

描述（由申请人提供）：许多致病菌表达感染性和发病机制所需的毒力基因。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细菌生长不是必需的，因此生物产生耐药性的选择压力应该更小。然而，针对大量的单个毒力因子使这种方法变得困难。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。抑制这些转录因子可能是发现抗生素的一种新的和通用的方法。虽然我们对细菌中转录的控制了解很多，但对那些与毒力有关的转录因子却知之甚少。一个例子是福氏志贺氏菌，其中VirF，一种arac型转录调节因子，负责所有下游毒力因子的表达，控制细胞内侵袭和细胞间扩散。志贺氏菌每年在全世界感染约1.65亿人，夺去100多万人的生命。虽然志贺氏菌病一直被认为是“第三世界”的一种疾病，但像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。基因敲除研究证实，抑制VirF表达足以阻断志贺氏菌在宿主体内的侵袭级联，从而增加对宿主免疫系统的易感性。我们这个项目的目标是双重的。首先，我们希望利用VirF作为我们的模型系统来证明靶向调节毒力基因表达作为一种新的抗生素方法的可行性。第二，进一步开发针对VirF的有效药物将导致针对志贺菌病的新型药物，这本身就是一个理想的目标，如上所述。这个R21应用程序的具体目标有两个。目标1:VirF检测方法的发展，以及高通量筛选（HTS）的一种或多种检测方法的适应。HTS将在密歇根大学化学基因组学中心的化学多样性化合物库（约15.5万+ 1.6万独特的天然产物提取物）上进行。目标2:HTS的“命中”将手动筛选潜在的“可药物性”属性。具有适当性质的hit将通过一些体外分析进一步表征。这些试验旨在验证命中，为后续先导优化提供指导，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，我们将开展“从成功到领先”的活动，包括模拟设计、合成和评估的迭代循环。我们还将研究这种鲜为人知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剂应用于许多人类病原体提供原理证明。该提案已被修订，以解决审稿人的意见，并包括我们在HTS检测开发方面取得的进展。

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.