Nucleobase Transport at the CNS Barriers

核碱基和核苷类似物被广泛用于治疗各种病毒感染和其他中枢神经系统(CNS)疾病。许多中枢神经系统靶向的碱基/核苷药物对大脑的渗透有限，这限制了它们的治疗效果。血脑屏障(BBB)和血-脑脊液屏障(脉络丛)的核碱基转运体可能在调节核碱/核苷类似物的中枢神经系统生物利用度中起关键作用。对中枢神经系统屏障核碱基转运机制的详细了解将有助于制定策略，以提高中枢神经系统靶向核碱基类似物的脑生物利用度，避免外周靶向核碱基药物的神经副作用。在血脑屏障和脉络丛中已经认识到了几种碱基转运系统；然而，潜在的碱基转运系统的分子身份和功能特征还没有被阐明。到目前为止，哺乳动物的碱基转运蛋白还没有被克隆出来。拟议的研究将集中在血脑屏障和脉络丛中核碱基转运体的分子鉴定和功能表征。其具体目标是：(1)为哺乳动物核碱基转运蛋白的克隆和鉴定开发克隆策略和表达系统。(2)克隆BBB的平衡碱基转运蛋白，并对其进行功能鉴定。(3)克隆和鉴定脉络丛钠离子依赖的碱基转运蛋白。我们将首先在酵母中开发新的互补克隆策略和合适的表达系统来克隆和鉴定哺乳动物的核碱基转运蛋白。然后，我们将使用我们的酵母策略从血脑屏障和脉络丛构建的cDNA文库中克隆核碱基转运体。一旦克隆，我们将在异源表达系统中表达这些碱基转运蛋白，并研究它们与生理碱基和临床上重要的碱基类似物相互作用的功能特征。还将探讨这些转运体在中枢神经系统屏障的分布情况。这些研究将极大地促进我们对碱基及其类似物在中枢神经系统屏障上的运输机制的理解。

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.