Live-Cell Fluorescence Lifetime FRET Assays for HTS

描述（由申请人提供）：该项目将以真正的高通量筛选（HTS）格式为小分子药物发现建立强大而通用的活细胞分析实施的概念验证。技术基础是荧光融合蛋白间FRET的荧光寿命（FLT）读数。为了克服传统荧光强度测量精度低的问题（在活细胞测定中尤其严重），需要在高温超导中进行寿命测量。然而，传统的寿命技术，即时间相关单光子计数（TCSPC），每个样品至少需要10秒才能获得足够的HTS精度。因此，无论是在微孔板读取器还是荧光寿命成像显微镜（FLIM）中进行，TCSPC对于实际的HTS来说都太慢了。我们的团队采用了一种全新的、创造性的方法，这主要依赖于由荧光创新公司开发的革命性的NovaFluor PR荧光寿命微孔板阅读器。NovaFluor采用直接波形记录（DWR），这是FI和明尼苏达大学托马斯研究小组最近合作开发的一种非常快速和精确的荧光寿命方法。DWR提供了与TCSPC相当的精度和分辨率，同时显著提高了数据采集的速度。在所有现有的荧光寿命方法中，只有DWR能同时提供有效高温超导所需的速度和精度。我们的另一项突破性创新是cell -and- wells （CNW）。在脉冲激光激发后，我们同时测量了微孔板中数百个细胞的反应，寿命读数提供的HTS数据与使用纯化蛋白靶标的任何基于强度的分析一样快，但精度和分辨率更高。目的1是在两个明确定义的测试系统上演示CNW方法，通过caspase-3切割标记肽和-synuclein的泛素化，以优化细胞处理，数据采集，仪器配置和数据还原的程序。目的2是开发一种针对重要蛋白靶点SERCA （sarco（endo）质网ca - atp酶）的高效检测方法，SERCA是所有哺乳动物细胞中钙调节的关键，在心力衰竭治疗中特别感兴趣。托马斯小组在开发SERCA光谱探针方面处于世界领先地位。目标3是使用LOPAC库进行第一次筛选。这项工作将为第二阶段更全面的化学空间探索奠定基础，导致FLT技术在药物发现方面的成功商业化。这个项目的意义在于，活细胞中的FLT有可能彻底改变HTS，从而大大改善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成功药物发现活动成为可能。荧光寿命在高温超导中的高潜在意义将使其成为一个高影响项目，即使在一期。

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.