Cell-Based Technology for Protein-Protein Interactions

描述(申请人提供)：人类基因组计划的完成为我们理解基因的功能和蛋白质在细胞通路中的作用提供了巨大的可能性。在活细胞中实时可视化和量化蛋白质-蛋白质复合体的方法将使基于生物学的药物发现成为可能。这些方法可能被用来阐明导致主要慢性疾病(包括癌症、心血管疾病、自身免疫和神经退行性疾病)的改变的细胞通路；将靶点映射到通路；对小分子文库进行高通量筛选；以及识别已知和未知药物的通路内和通路外效应。现有的用于可视化和量化活细胞中蛋白质-蛋白质相互作用的分析方法(如蛋白质片段互补分析，PCA)代表着朝着基于途径的药物发现迈出的一步。这种分析的一个主要缺点是使用大报告荧光蛋白，这些蛋白可能会干扰和/或立体地阻碍目标蛋白的功能，或者产生假阳性或阴性结果。使用大报告蛋白的固有复杂性使得这种检测的设计、开发和验证既耗时又昂贵，限制了这项技术的应用和影响。为了克服与现有方法相关的技术困难，我们将开发一种新的方法来在体内测量蛋白质-蛋白质复合体，该方法检测来自已被定位并生物合成地结合到目标蛋白质中的小荧光团的信号。我们将建立表达正交氨酰基转移酶/琥珀抑制物tRNA对的细胞系，该tRNA对在目标mRNA中由琥珀终止密码子指定的位置将BODIPY-氨基酸衍生物特异性地掺入延长的目标蛋白序列中。荧光偏振测量将用于检测荧光团标记的目标蛋白与第二种蛋白的相互作用。在第一阶段的研究中，我们将建立一种基于细胞的分析方法，用于测量FK506介导的FKBP12与钙调神经磷酸酶(CN)的二聚化反应。目的1建立FKBP12和CN在哺乳动物细胞中的表达。在目标2中，我们将对一系列BODIPY-FKBP12的表达进行优化，并对其荧光偏振特性进行表征。在目标3中，我们将优化BODIPY-FKBP12和CN的共表达，并确定哪些BODIPY-FKBP12蛋白最适合用荧光偏振来检测FK506介导的二聚化。我们相信，我们的方法将允许最终用户快速开发基于细胞的分析方法，用于量化蛋白质-蛋白质相互作用，可用于绘制细胞内路径图和筛选小分子蛋白质-蛋白质相互作用调节剂。

DESCRIPTION (provided by applicant): The completion of the Human Genome Project presents us with the tremendous possibility of understanding the function of genes and the role of proteins in cellular pathways. Methods for visualizing and quantifying protein-protein complexes in real time in living cells would enable biology-based drug discovery on a large scale. These methods could potentially be used to elucidate altered cellular pathways that underlie major chronic diseases including cancer, cardiovascular, autoimmune and neurodegenerative conditions; to map targets into pathways; to perform high-throughput screening of small-molecule libraries; and to identify on- pathway and off-pathway effects of known and unknown drugs. Existing assays (e.g. Protein fragment Complementation Assay, PCA) used for visualization and quantification of protein-protein interactions in live cells represent a step toward pathway-based drug discovery. A major drawback of such assays is the use of large reporter fluorescent proteins that may interfere with and/or sterically hinder the target protein's function or generate false positive or negative results. Inherent complexities with use of large reporter proteins make the design, development and validation of such assays time-consuming and expensive, limiting the application and impact of this technology. To overcome the technical difficulties associated with existing approaches, we will develop a novel method for in vivo measurement of protein-protein complexes that detects signals from a small fluorophore that has been site-specifically and biosynthetically incorporated into the protein of interest. We will generate cell lines that express an orthogonal aminoacyl transferase/amber suppressor tRNA pair that specifically incorporates a BODIPY-amino acid derivative into the elongating target protein sequence at positions in the target mRNA designated by the amber stop codon. Fluorescence polarization measurements will be used to detect the interaction of the fluorophore-labeled target protein with a second protein. In the Phase I studies, as a model assay we will establish a cell-based assay for measuring the FK506-mediated dimerization of FKBP12 with calcineurin (CN). In Aim 1 we will establish the expression of FKBP12 and CN in mammalian cells. In Aim 2, we will optimize the expression and characterize the fluorescence polarization of a series of BODIPY-FKBP12. In Aim 3, we will optimize co-expression of BODIPY-FKBP12 and CN and determine which BODIPY-FKBP12 proteins are optimal for measuring FK506-mediated dimerization using fluorescence polarization. We believe our approach will allow the end-user to rapidly develop cell-based assays for quantifying protein-protein interactions that can be used to map intracellular pathways and screening of small-molecule protein-protein interaction modulators.