Analysis of Intracellular Drug Sequestration

细胞内药物封存分析

基本信息

批准号：
7534009
负责人：
JEFFREY P KRISE
金额：
$ 20.55万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-01-01 至 2010-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Drug targets are typically confined to a single organelle population within a cell. Likewise, drugs themselves often have the tendency to selectively accumulate within specific cellular compartments. Our research is focused on understanding driving forces important in the transport of small molecule type drugs within the array of intracellular compartments associated with mammalian cells. We aim to use this understanding to rationally develop novel drug delivery strategies to maximize the interaction of a drug with its target in the context of a single cell. The goal of the research outlined in this application is to understand mechanisms that are involved in the sequestration of anti-cancer drugs into cytoplasmic organelles of multi-drug resistant (MDR) cancer cell lines and is a process that does not seem to occur in drug sensitive cells. This sequestration has been shown to cause drastic reductions in the amount of drug available to partition into cellular compartments housing drug targets. For experiments outlined in this application, we have acquired three pairs of human leukemic cell lines. Each pair is comprised of a drug sensitive line and its corresponding MDR variant created through drug selection. We have shown that each MDR variant is able to sequester the anticancer drug, daunorubicin, into distinct cytoplasmic organelles. The first segment of this application is focused on identifying drug sequestering organelle(s). The sequestration will be assessed using a series of fluorescent molecules with different physicochemical properties, which will broadly represent different categories of anticancer agents. Drug sequestering organelles will be identified using fluorescence co-localization studies with organelle specific fluorescent probes and vital stains. The second phase of this application describes methods used to reveal the fundamental mechanisms involved in drug sequestration. Comparative proteomic approaches will be used to determine differences in protein expression between drug sequestering organelles isolated from MDR cells and the same organelles isolated from drug sensitive cells. To confirm their involvement in drug sequestration, identified proteins will be specifically down-regulated in MDR cells, and the sequestration capacity will be reevaluated. The final phase of this application will examine the substrate specificity for sequestration in identified organelles. We will utilize novel quantitative assays to measure drug accumulation into sequestering organelles. Using these assays, structure transport relationships will be designed in order to identify the key structural requirements that are essential for drug sequestration. Together these studies will give us a fundamental understanding of this drug sequestration phenotype. This understanding will guide us in the development of novel drug design/modification strategies that will be useful in producing drugs with reduced sequestration tendency and, therefore, an improved capacity to interact with relevant target sites within MDR cancer cells.

描述（由申请人提供）：药物靶标通常仅限于细胞内的单个细胞器种群。同样，药物本身通常具有选择性积累在特定的细胞室内的趋势。我们的研究集中在理解对与哺乳动物细胞相关的细胞内隔室中小分子型药物运输重要的驱动力。我们的目的是利用这种理解来合理发展新型的药物输送策略，以最大化药物与单个细胞的相互作用。本应用中概述的研究的目的是了解抗癌药物隔离为多药耐药（MDR）癌细胞系的细胞质细胞器中涉及的机制，并且似乎在药物敏感细胞中似乎不存在。该固存已被证明会导致可用于分配到壳体药物靶标的细胞室的药物量的急剧减少。对于此应用中概述的实验，我们获得了三对人类白血病细胞系。每对均由药物敏感线及其通过药物选择产生的相应的MDR变体组成。我们已经表明，每个MDR变体都能够将抗癌药物Daunorubicin隔离为不同的细胞质细胞器。该应用的第一部分集中在识别药物隔离细胞器上。将使用具有不同物理化学特性的一系列荧光分子来评估隔离，该分子将广泛代表不同类别的抗癌药物。使用细胞器特异性荧光探针和重要污渍的荧光共定位研究，将确定药物隔离细胞器。该应用程序的第二阶段描述了用于揭示药物隔离涉及的基本机制的方法。比较蛋白质组学方法将用于确定从MDR细胞分离的药物隔离细胞器与从药物敏感细胞分离的相同细胞器中的蛋白质表达差异。为了确认它们参与药物固醇，将在MDR细胞中特别下调鉴定的蛋白质，并将重新评估隔离能力。该应用程序的最后阶段将检查已识别细胞器中隔离的底物特异性。我们将利用新颖的定量测定法测量药物积累到隔离细胞器中。使用这些测定法，将设计结构传输关系，以确定对药物隔离至关重要的关键结构要求。这些研究将共同使我们对这种药物隔离表型有基本的了解。这种理解将指导我们开发新的药物设计/修饰策略，这些策略将有助于生产具有较低的隔离趋势的药物，因此可以提高与MDR癌细胞中相关目标位点相互作用的能力。