Nucleobase Transport at the CNS Barriers

中枢神经系统屏障的核碱基运输

• 批准号: 6923592
• 负责人: Joanne Wang
• 金额: $ 21.63万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923592
• 关键词: blood brain barrier; choroid plexus; genetic library; homeostasis; in situ hybridization; ion transport; laboratory rabbit; laboratory rat; membrane permeability; membrane transport proteins; molecular cloning; northern blottings; nucleobase; yeasts

Nucleobase and nucleoside analogs are widely used in the treatment of various viral infections and other diseases in the central nervous system (CNS). Many CNS targeted nucleobase/nucleoside drugs exhibit limited penetration into the brain, which limits their therapeutic effectiveness. Nucleobase transporters at the blood brain barrier (BBB) and the blood- cerebrospinal fluid barrier (choroid plexus) may play a critical role in governing the CNS bioavailability of nucleobase/nucleoside analogs. A detailed understanding of the mechanisms of nucleobase transport at the CNS barriers will allow the development of strategies to enhance the brain bioavailability of CNS targeted nucleobase analogs and to avoid neurological side effects of peripherally targeted nucleobase drugs. Several nucleobase transport systems have been recognized at the BBB and choroid plexus; however, the molecular identity and functional characteristics of the underlying nucleobase transporters have not been elucidated. To date, mammalian nucleobase transporters have not been cloned. The proposed studies will focus on molecular identification and functional characterization of nucleobase transporters in the BBB and choroid plexus. The specific aims are: (1) Develop cloning strategies and expression systems for cloning and characterization of mammalian nucleobase transporters. (2) Clone and functionally characterize the equilibrative nucleobase transporter from the BBB. (3) Clone and functionally characterize the Na+-dependent nucleobase transporter from the choroid plexus. We will first develop novel complementation cloning strategies in yeast and suitable expression systems for cloning and characterization of mammalian nucleobase transporters. We will then clone the nucleobase transporters from cDNA libraries constructed from the BBB and choroid plexus using our yeast strategy. Once cloned, we will express these nucleobase transporters in heterologous expression systems and investigate their functional characteristics in interacting with physiologic nucleobases and clinically important nucleobase analogs. The distribution of these transporters at the CNS barriers will also be explored. These studies will greatly advance our understanding of the transport mechanisms of nucleobases and their analogs at the CNS barriers.

核苷酸酶和核苷类似物广泛用于治疗中枢神经系统（CNS）中各种病毒感染和其他疾病。 许多中枢神经系统靶向核苷酶/核苷药物都显示出有限的渗透到大脑中，从而限制了它们的治疗效果。 血液脑屏障（BBB）和血液脊髓液屏障（脉络膜丛）处的核碱酶转运蛋白在管理核苷酶/核苷类似物的CNS生物利用率方面可能起关键作用。 对中枢神经系统屏障处核碱酶转运机制的详细理解将使策略的发展能够增强中枢神经系统靶向核苷酶类似物的脑生物利用度，并避免外周靶向核碱性酶药物的神经副作用。 在BBB和脉络丛上已经识别出几种核碱基转运系统。然而，尚未阐明基础核碱基转运蛋白的分子身份和功能特征。 迄今为止，尚未克隆哺乳动物核碱酶转运蛋白。 拟议的研究将重点介绍BBB和脉络丛中核酶转运蛋白的分子鉴定和功能表征。 具体目的是：（1）开发克隆策略和表达系统，用于克隆和表征哺乳动物核碱基转运蛋白。 （2）克隆并在功能上表征来自BBB的平衡性核酶转运蛋白。 （3）克隆并在功能上表征来自脉络丛的Na+依赖性核酶转运蛋白。 我们将首先在酵母中开发新型的互补克隆策略，以及适当的表达系统，用于克隆和表征哺乳动物核苷酶转运蛋白。 然后，我们将使用我们的酵母策略从由BBB和脉络膜丛构建的cDNA文库中克隆核碱基转运蛋白。 克隆后，我们将在异源表达系统中表达这些核酶转运蛋白，并研究其在与生理核苷酸酶相互作用和临床上重要的核酶类似物中相互作用时的功能特征。 这些转运蛋白在中枢神经系统屏障的分布也将被探索。 这些研究将大大提高我们对中枢神经系统障碍物核碱基及其类似物的运输机制的理解。