HTS assay development for bacterial transcription factors

描述（由申请人提供）：迫切需要新的抗生素来治疗细菌感染。该提案的目标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，这些抑制剂将作为开发新抗生素的先导化合物。存在许多已知的TF的实例，其是致病性细菌物种的毒力所需的。此外，大多数细菌TF的作用机制非常清楚。因此，TF代表了抗菌治疗的优良靶标。我们选择了两个TF，环AMP受体蛋白（CRP）和SoxS，这是所需的多个细菌物种的毒力和/或抗生素耐药性。我们假设CRP或SoxS的化学抑制剂将是有效的抗菌剂，抑制毒力或抵消多种重要病原体的抗生素耐药性;目前没有这样的抑制剂。此外，我们开发的HTS方法将作为一个通用系统，用于识别任何细菌TF的抑制剂。我们将开发HTS检测方法，提供可重现的，低噪声的稳健信号。这些将基于先前开发的原理验证测定，包括用于CRP功能的生物发光测定，其给出高Z'评分（在96孔格式中>0.86）。CRP和SoxS的独立HTS检测试剂盒将作为彼此的对照品，无需进行二次筛查。我们将通过使用小的化合物库进行筛选来测试这些测定。这将确定屏幕对HTS的适用性， 将决定它们是否适合小型化。我们将使用一组成熟的体内和体外试验验证和表征推定的CR和SoxS抑制剂。我们期望开发出可应用于HTS的用于CRP和SoxS功能的稳健的、高度可重复的检测方法。一旦验证，检测试剂盒将提交给HTS的分子库筛选中心网络（MLSCN）。CRP和SoxS抑制剂，我们希望在这项工作中确定，并在未来的HTS筛选代表的先导化合物在新的抗菌药物的发展。在未来的工作中，我们将使用鼠伤寒沙门氏菌（一种每年在美国造成数百万感染的细菌）和鼠疫耶尔森氏菌（鼠疫）（一种主要的生物恐怖主义威胁）的体外和体内感染模型来测试这些化合物的功效。

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.