Fluorescent Probes for Hydrophobic Metabolites(RMI)

描述（由申请人提供）：本研究的目的是阐明细胞内和细胞外的代谢途径。在本应用中，我们将开发能够测定细胞外液和细胞内液中各种疏水代谢物浓度的荧光探针。探针将通过荧光标记脂肪酸结合蛋白（FABP）突变体构建。这些探针的一个独特之处在于它们能够测量未结合（水单体）疏水代谢物的浓度，从而提供对代谢途径的分子成分的直接测量。本工作的重点将是开发用于测定游离脂肪酸（FFAu）浓度的探针。在健康方面，循环中的游离脂肪酸受到高度调控，多达40种不同种类的游离脂肪酸的分布（剖面）也受到高度调控。FFA具有多种基本功能；它们提供了人体能量的主要部分，是重要的信号分子。在糖尿病、心脏和脑缺血以及可能的癌症等疾病中，即使是游离脂肪酸水平和分布的微小变化，也可能对人体健康造成严重危害。由于技术上的限制，对FFA剖面的准确监测非常具有挑战性。FFA以及大多数疏水代谢物通过未结合（水相）分子介导其活性。然而，除了我们正在开发的FFAu探针外，几乎没有方法可以准确测定细胞外液和细胞内液中的未结合分子。在这些研究中，我们将：1)使用高通量系统（HTS）生成和筛选针对单个FFAu的40个探针，这些探针在初步研究中产生的探针具有与ADI FAB显著不同的FFA结合谱；2)开发方法来确定含有FFAu混合物的细胞外液中单个FFAu的浓度；3)开发方法来确定活细胞中不同FFAu的细胞内浓度。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate metabolic pathways in both the intra- and extracellular regime. In this application we will develop fluorescent probes capable of determining the concentrations of a wide range of hydrophobic metabolites in both extra and intracellular fluids. Probes will be constructed by fluorescent labeling mutants of fatty acid binding proteins (FABP). A unique feature of these probes is they enable the measurement of the concentration of unbound (aqueous monomers) hydrophobic metabolites, thereby providing direct measurements of the molecular constituents of the metabolic pathways. The focus of this work will be the development of probes for determining the concentration of unbound free fatty acids (FFAu). In health, circulating FFA are highly regulated as is the distribution (profile) of the as many as 40 different species of FFA. FFA serve a variety of essential functions; they provide a major portion of the body's energy and are important as signaling molecules. Even modest changes in the levels and distributions of FFA that may occur in diseases such as diabetes, cardiac and cerebral ischemia and probably cancer, can be highly deleterious to human health. Because of technical limitations accurate monitoring of FFA profiles has been very challenging. FFA as well as most hydrophobic metabolites mediates their activities through the unbound (aqueous phase) molecules. However, with the exception of the probes we are developing for FFAu there are virtually no methods for the accurate determination of the unbound molecules in extracellular as well as intracellular fluids. In these studies we will: 1) generate a suite of 40 probes specific to individual FFAu using a high throughput system (HTS) for generating and screening new probes that in preliminary studies has yielded probes with FFA binding profiles that are significantly different from those of ADI FAB, 2) develop methods to determine the concentration of individual FFAu in extracellular fluids containing mixtures of FFAu and 3) develop methods to determine intracellular concentrations of different FFAu in living cells.