HTS assay development for bacterial transcription factors

描述(申请人提供)：迫切需要新的抗生素来治疗细菌感染。这项建议的目标是开发一种高通量筛选(HTS)方法来确定细菌转录因子(TF)的小分子抑制剂，这些小分子抑制剂将作为新抗生素开发中的先导化合物。有许多已知的例子是致病细菌物种毒力所必需的转铁蛋白。此外，大多数细菌转录因子的作用机制是非常清楚的。因此，TF是抗菌治疗的极佳靶点。我们选择了两个转录因子，环-AMP受体蛋白(CRP)和SOXS，它们是多种细菌毒力和/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剂，可以抑制多种重要病原体的毒力或对抗抗生素耐药性；目前还没有这样的抑制剂。此外，我们开发的HTS方法将作为一个通用系统来识别任何细菌转铁蛋白的抑制剂。我们将开发HTS检测方法，提供可重现的、低噪声的强健信号。这些将基于以前开发的原理验证分析，包括给予高Z‘分数(96孔格式为0.86)的CRP功能的生物发光分析。针对CRP和SOXS的独立HTS检测将作为彼此的对照，消除了对二次筛查的需要。我们将通过使用小型化合物库进行筛选来测试这些分析。这将确立屏幕对HTS的适用性，以及 将决定它们是否适合小型化。我们将使用一系列成熟的体内和体外试验来验证和表征推定的CR和SOXS抑制剂。我们期望开发出可靠的、高重复性的CRP和SOXS功能的检测方法，可以应用于HTS。一旦得到验证，检测结果将提交给HTS的分子图书馆筛选中心网络(MLSCN)。我们希望在这项工作和未来的HTS筛选中确定的CRP和SOXS抑制剂代表着开发新型抗菌剂的先导化合物。在未来的工作中，我们将使用鼠伤寒沙门氏菌和鼠疫耶尔森菌(鼠疫)的体外和体内感染模型来测试这些化合物的有效性。鼠伤寒沙门氏菌是一种每年在美国造成数百万感染的细菌，鼠疫是一种主要的生物恐怖主义威胁。

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.