HIGH CONTENT SCREENING FOR EPITHELIAL BIOLOGY

描述（由申请人提供）：BioTechPlex拟开发一种高含量、高通量、基于细胞的筛选系统，用于使用上皮细胞进行药物发现。该系统将使多个细胞荧光团可报告的分子物种的动态响应能够在96孔板上同时测量。动态响应将在与各自细胞内动力学过程的时间性质相一致的时间框架内测量。目标位点的激活或抑制将根据三个荧光探针的动态“指纹”的表征和分类，以高特异性确定。在第一阶段，BioTechPlex开发了多荧光声光板读取器（MAPR(tm)）系统的光学和固件。MAPR同时测量来自96个细胞样品的三种荧光信号，每孔停留时间为10 ms。在第二阶段，BioTechPlex将进一步开发MAPR，增加生物信息学数据库，人工智能决策和验证整个系统的制定。这将通过实现以下3个目标来实现。1)在MAPR中加入流体处理和环境调节，包括机器人板和液体处理以及温度和大气调节。2)开发目标识别算法，对三种荧光团动态的唯一标识符进行分类、表征和归档，并提取这些信息用于目标识别和决策标记。3)在干扰毒毒碱、肾上腺素能和IPS受体激活的信号转导途径之前和之后，使用正常的、病理的和工程的呼吸上皮细胞系验证完整的MAPR系统，这些信号转导途径可预测地干扰细胞内离子，从而扰乱质膜电位。这项技术将为制药行业提供一种新颖的、基于多探针细胞的平台，用于药物发现、验证和细胞毒性的高含量筛选和高通量药物筛选。该系统满足FDA的需求（由FDA资助）。此外，该项目直接响应了2004年发布的NIH路线图倡议。该项目也直接响应了NCRR在2005年SBIR中对高通量筛选技术的开发，以及NIDDK对上皮生物学的普遍兴趣。

DESCRIPTION (provided by applicant): BioTechPlex proposes to develop a high content, high throughput, cell-based screening system for drug discovery using epithelial cells. This system will enable the dynamic responses of multiple cellular fluorophore-reportable molecular species to be simultaneously measured in a 96 well plate. The dynamic responses will be measured in a time frame consistent with the temporal nature of the respective intracellular kinetic processes. The activation or inhibition of a target site will be determined with high specificity following the characterization and classification of the dynamic "fingerprint" from three fluorescent probes. In Phase I, BioTechPlex developed the optics and the firmware of the Multi-Fluorophore Acousto Optic Plate Reader (MAPR(tm)) system. MAPR simultaneously measures three fluorescent signals from 96 cell samples with a dwell time of 10 ms per well. In Phase II, BioTechPlex will further develop MAPR, add bioinformatics data base, Al decision making and validate the entire system making. This will be achieved by accomplishing the following 3 objectives. 1) Add fluid handling and environmental conditioning to MAPR by incorporating robotic plate and liquid handling as well as temperature and atmospheric regulation. 2) Develop Target Identification Algorithms that will classify, characterize and file the unique identifiers of the dynamics of three fluorophores as well as extract this information for target identification and decision marking. 3) Validate the complete MAPR system using normal, pathologic and engineered respiratory epithelial cell lines prior to and following perturbation of muscarinic, adrenergic and IPS receptor activated signal transduction pathways which predictably perturb intracellular ions and thus plasma membrane potential. This enabling technology will provide the pharmaceutical industry a novel, multi-probe cell-based platform for high content screening and high-throughput drug screening for drug discovery, validation, and cytotoxicity. This system addresses the needs of FDA (funded by FDA). In addition, the project directly responds to the NIH Roadmap Initiatives released in 2004. This project also directly responds to the 2005 SBIR general solicitations by NCRR for the development of high throughput screening technologies as well as by NIDDK for general interest in epithelial biology.