BLOOD-BRAIN TRANSPORTERS OF ANTIVIRAL NUCLEOSIDES

几个核苷类似物具有抗逆转录病毒活性， 目前正在使用或正在积极考虑治疗艾滋病 脑病。维持核苷传递和维持核苷释放的机制 脑细胞外液的动态平衡被认为包括 血脑屏障和血脑脊液屏障的转运体。这个 这项研究项目的长期目标是描述 抗病毒核苷(和天然的)的分子过程 发生的核苷)通过细胞运输通过 构成血脑屏障的细胞(内皮细胞)和 血脑脊液屏障(脉络丛上皮细胞)。这些 目标包括对分子身份的理解 转运蛋白，转运蛋白所在的膜位置，以及 底物和缓蚀剂的动力学传输特性 专一性。在这项提议中，大脑的核苷转运体 血管内皮细胞和脉络丛将通过 核苷转运缺陷酵母(S. 微血管和脉络丛基因转化酿酒酵母) 图书馆。钠无关者和钠-的代表 将对依赖的核苷转运体进行选择和测序。 针对所推导的氨基酸序列的抗体 转运体将被准备并用于确定他们的细胞 利用光和超微结构进行定位和相对丰度 免疫细胞化学。钠依赖的核苷转运体将 在杆状病毒表达系统中的表达及其抗病毒作用 由电生理决定的运输特性 技巧。这些研究的结果将有助于开发 A)优化抗艾滋病毒药物脑内给药的策略，b) 测定潜在的抗病毒核苷的亲和力 核苷转运体的快速体外方法，以及c)通过 构效关系在抗病毒核苷转运中的应用 有利于药物输送和抗病毒功效的特性。

Several nucleoside analogs have antiretroviral activity and are currently in use or under active consideration for treatment of AIDS encephalopathy. The mechanisms that maintain nucleoside delivery and homeostasis in brain extracellular fluids is believed to involve transporters at the blood-brain barrier and the blood-CSF barrier. The long-term goals of this research project are to characterize the molecular process by which antiviral nucleosides (and naturally occurring nucleosides) are transported transcellularly through the cells comprising the blood-brain barrier (endothelial cells) and the blood-CSF barrier (epithelial cells of the choroid plexus). These goals include an understanding of the molecular identity of the transporters, the membrane sites where transporters are located, and the kinetic transport characteristics of substrate and inhibitor specificity. In this proposal, nucleoside transporters of the brain endothelial cell and choroid plexus will be identified by complementation cloning of nucleoside transport-deficient yeast (S. cerevisiae) transformed with microvessel and choroid plexus cDNA libraries. Representatives of the sodium-independent and sodium- dependent nucleoside transporters will be selected and sequenced. Antibodies specific to the deduced amino acid sequences of the transporters will be prepared and used to determine their cellular location and relative abundance using light and ultrastructural immunocytochemistry. A sodium-dependent nucleoside transporter will be expressed in a baculovirus expression system and its antiviral transport characteristics determined by electrophysiological techniques. The results of these studies will be useful for developing strategies for a) optimizing anti-HIV drug delivery to the brain, b) determining the affinity of potential antiviral nucleosides for nucleoside transporters by rapid in vitro methods, and c) designing by structure-activity approaches new antiviral nucleosides with transport properties that favor drug delivery and antiviral efficacy.