Live-Cell Fluorescence Lifetime FRET Assays for HTS

描述（由申请人提供）：该项目将建立一个强大的和多功能的实施活细胞测定的概念验证，在一个真正的高通量筛选（HTS）格式的小分子药物发现。技术基础是荧光融合蛋白之间FRET的荧光寿命（FLT）读出。在HTS中需要寿命测量以克服常规荧光强度测量的低精度，这在活细胞测定中特别严重。然而，传统的寿命技术，即，时间相关单光子计数（TCSPC）每个样本至少需要10秒才能获得足够的HTS精度。因此，无论是在酶标仪中还是在荧光寿命成像显微镜（FLIM）中进行，TCSPC对于实际HTS来说都太慢了。我们的团队采用了一种全新的创造性方法，该方法主要依赖于Fluorescence Innovations开发的革命性NovaFluor PR荧光寿命酶标仪。NovaFluor采用直接波形记录（DWR），这是一种非常快速和精确的荧光寿命方法，最近由FI和明尼苏达大学的托马斯研究小组合作开发。DWR提供了与TCSPC相当的精度和分辨率，同时大大提高了数据采集速度。在所有现有的荧光寿命方法中，只有DWR提供了有效HTS所需的速度和精度。我们的另一项突破性创新是Cells-and-威尔斯（CNW）。在用脉冲激光激发后，我们同时测量微孔板孔中数百个细胞的响应，并且寿命读出提供与使用纯化蛋白质靶的任何基于强度的测定一样快的HTS数据，但具有更好的精度和分辨率的数量级。目的1是证明CNW方法上两个定义明确的测试系统，裂解的标记肽的caspase-3和泛素化的-突触核蛋白，以优化程序的细胞处理，数据采集，仪器配置，和数据减少。目的2是开发一种用于重要蛋白质靶点SERCA（肌浆网Ca-ATP酶）的高性能测定方法，该蛋白质靶点是所有哺乳动物细胞中钙调节的关键，并且在心力衰竭治疗中特别感兴趣。托马斯集团在SERCA光谱探针的开发方面处于世界领先地位。目标3是用LOPAC文库进行首过筛选。这项工作将为第二阶段更全面的化学空间探索奠定基础，从而使FLT技术成功商业化用于药物发现。该项目的重要性源于FLT在活细胞中彻底改变HTS的明确潜力，从而大大改善了药物发现过程的投入。我们设想，我们的方法将使目前只能通过荧光强度筛选的广泛靶点和系统的药物发现活动取得成功。高温超导体中荧光寿命的高潜在意义将使其成为一个高影响力的项目，即使在第一阶段。

DESCRIPTION (provided by applicant): This project will establish proof-of-concept for a powerful and versatile implementation of live-cell assays in a true high-throughput screening (HTS) format for small-molecule drug discovery. The technological basis is fluorescence lifetime (FLT) readout of FRET between fluorescent fusion proteins. Lifetime measurement is needed in HTS to overcome the low precision of conventional fluorescence intensity measurements, which is particularly severe in live-cell assays. However, conventional lifetime technology, i.e., time-correlated single-photon counting (TCSPC), takes at least 10 seconds per sample to obtain adequate precision for HTS. Thus, whether carried out in a microplate reader or in fluorescence lifetime imaging microscopy (FLIM), TCSPC is much too slow for practical HTS. Our team has taken an entirely fresh and creative approach, which critically relies on the revolutionary NovaFluor PR fluorescence lifetime microplate reader developed by Fluorescence Innovations. NovaFluor employs Direct Waveform Recording (DWR), an exceptionally fast and precise fluorescence lifetime method recently developed in collaboration between FI and the Thomas research group at the University of Minnesota. DWR provides precision and resolution equivalent to TCSPC while dramatically increasing the speed of data acquisition. Of all the existing fluorescence lifetime methods, only DWR offers both the speed and precision needed for effective HTS. Our other breakthrough innovation is Cells-and-Wells (CNW). We simultaneously measure the response of hundreds of cells in a microplate well, after excitation with a pulsed laser, and the lifetime readout provides HTS data as fast as any intensity-based assay employing purified protein targets, but with an order of magnitude better precision and resolution. Aim 1 is to demonstrate the CNW method on two well-defined test systems, cleavage of a labeled peptide by caspase-3 and ubiquitination of -synuclein, in order to optimize procedures in cell handling, data acquisition, instrument configuration, and data reduction. Aim 2 is to develop a high-performance assay for an important protein target, SERCA, the sarco(endo)plasmic reticulum Ca-ATPase, which is key to calcium regulation in all mammalian cells, and of particular interest in heart failure therapies. The Thomas group leads the world in developing spectroscopic probes of SERCA. Aim 3 is to conduct a first-pass screening with the LOPAC library. This work will set the stage for a more comprehensive exploration of chemical space in Phase II, leading to successful commercialization of FLT technology for drug discovery. The significance of this project stems from the clear potential of FLT in live cells to revolutionize HTS, resulting in a vastly improved input into the drug discovery process. We envision our approach will enable successful drug discovery campaigns for a wide range of targets and systems that currently can only be screened by fluorescence intensity. The high potential significance of fluorescence lifetime in HTS will make this a high-impact project, even in Phase I.