HTS assay development for bacterial transcription factors

细菌转录因子的 HTS 检测开发

• 批准号: 8739660
• 负责人: Joseph Thomas Wade
• 金额: $ 19万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-24 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8739660
• 关键词: Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Biological Assay; Bioluminescence; Bioterrorism; Cells; Chemicals; Collection; Cyclic AMP Receptor Protein; Development; Future; Generic Drugs; Goals; In Vitro; Infection; Lead; Libraries; Life; Miniaturization; Modeling; Molecular Bank; Noise; Plague; Play; Proteins; Reporter; Salmonella typhimurium; Signal Transduction; System; Testing; Virulence; Work; Yersinia pestis; antimicrobial; assay development; base; efficacy testing; high throughput screening; in vitro Assay; in vivo; inhibitor/antagonist; luminescence; novel; pathogen; protein function; public health relevance; screening; small molecule; transcription factor

DESCRIPTION (provided by applicant): There is a desperate need for new antibiotics to treat bacterial infections. The goal of this proposal is to develop a High-Throughput Screening (HTS) approach to identify small molecule inhibitors of bacterial Transcription Factors (TFs) that will serve as lead compounds in the development of new antibiotics. There are many known examples of TFs that are required for the virulence of pathogenic bacterial species. Furthermore, the mechanism of action of most bacterial TFs is extremely well understood. Hence, TFs represent excellent targets for antibacterial therapy. We have selected two TFs, cyclic-AMP receptor protein (CRP) and SoxS, which are required for the virulence and/or antibiotic resistance of multiple bacterial species. We hypothesize that chemical inhibitors of CRP or SoxS will be effective antimicrobials that inhibit virulence or counteract antibiotic resistance of multiple important pathogens; no such inhibitors are currently available. Furthermore, the HTS approach we develop will serve as a generic system for identifying inhibitors of any bacterial TF. We will develop HTS assays that give reproducible, robust signal with low noise. These will be based on previously-developed proof-of-principle assays, including a bioluminescence assay for CRP function that gives a high Z' score (>0.86 in 96-well format). The independent HTS assays for CRP and SoxS will serve as controls for each other, eliminating the need for secondary screens. We will test these assays by performing screens with small libraries of compounds. This will establish the applicability of the screens to HTS, and will determine their suitability for miniaturization. We will validate and characterize putative CR and SoxS inhibitors using a battery of well-established in vivo and in vitro assays. We expect to develop robust, highly reproducible assays for CRP and SoxS function that can be applied to HTS. Once validated, the assays will be submitted to the Molecular Libraries Screening Centers Network (MLSCN) for HTS. The CRP and SoxS inhibitors we expect to identify in this work and in future HTS screening represent lead compounds in the development of novel antimicrobials. In future work we will test the efficacy of these compounds using in vitro and in vivo infection models for Salmonella Typhimurium, a bacterium responsible for millions of infections annually in the US, and Yersinia pestis (plague), a major bioterrorism threat.

描述（由申请人提供）：迫切需要新的抗生素来治疗细菌感染。该提案的目的是开发一种高通量筛选（HTS）方法，以鉴定细菌转录因子（TF）的小分子抑制剂（TFS），该抑制剂将用作开发新抗生素的铅化合物。有许多已知的TF的例子是致病细菌物种的毒力所必需的。此外，大多数细菌TFS的作用机理都非常了解。因此，TFS代表了抗菌治疗的极好靶标。我们选择了两种TF，Cyclim Amp受体蛋白（CRP）和SOX，这是多种细菌物种的毒力和/或抗生素耐药性所必需的。我们假设CRP或Soxs的化学抑制剂将是有效的抗菌药物，可抑制多种重要病原体的毒力或抵消抗生素耐药性；目前没有这样的抑制剂。此外，我们开发的HTS方法将作为识别任何细菌TF抑制剂的通用系统。我们将开发HTS测定法，该测定法具有低噪声的可重现，可靠的信号。这些将基于先前开发的原则检验，包括用于CRP功能的生物发光测定，该测定具有较高的Z'分数（以96孔格式> 0.86）。 CRP和SOX的独立HTS分析将彼此充当控制，从而消除了对次级筛选的需求。我们将通过用小型化合物库进行屏幕来测试这些测定法。这将确定屏幕对HTS的适用性，以及 将确定其适合小型化的能力。我们将使用一系列良好的体内和体外测定法来验证和表征推定的CR和SOX抑制剂。我们期望可以针对可应用于HTS的CRP和SOXS功能开发可重现的高度可重现测定法。一旦验证，该测定将提交给HTS的分子库筛选中心网络（MLSCN）。我们期望在这项工作中鉴定出的CRP和SOX抑制剂，并且在未来的HTS筛选中代表了新型抗菌剂的发展中的铅化合物。在未来的工作中，我们将使用体外和体内感染模型来测试这些化合物的疗效，沙门氏菌伤寒沙门氏菌，该细菌每年在美国和耶尔森氏菌（Pestis）（PESTIS）（PESTIS）（PESTIS）（一种主要的生物恐怖主义威胁）。