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。本提案的目标是开发一种高通量筛选（HTS）方法来鉴定细菌转录因子（TFs）的小分子抑制剂，这些小分子抑制剂将作为开发新抗生素的先导化合物。有许多已知的致病细菌物种的毒力所需要的tf的例子。此外，大多数细菌tf的作用机制已经非常清楚。因此，tf是抗菌治疗的极佳靶点。我们选择了两种TFs，环amp受体蛋白（CRP）和SoxS，它们是多种细菌的毒力和/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剂，可以抑制多种重要病原体的毒力或抵消抗生素耐药性；目前还没有这种抑制剂可用。此外，我们开发的HTS方法将作为鉴定任何细菌TF抑制剂的通用系统。我们将开发HTS分析，提供可重复的、鲁棒的低噪声信号。这些将基于先前开发的原理验证分析，包括CRP功能的生物发光分析，其Z′评分很高（96孔格式>0.86）。CRP和SoxS的独立HTS检测将相互作为对照，从而消除了二次筛选的需要。我们将通过对小化合物库进行筛选来测试这些分析。这将确定屏幕对HTS的适用性

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.