BLOOD-BRAIN TRANSPORTERS OF ANTIVIRAL NUCLEOSIDES

几种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用于治疗艾滋病 脑病 维持核苷递送的机制和 脑细胞外液中的稳态被认为涉及 在血脑屏障和血-CSF屏障的转运蛋白。 的 该研究项目的长期目标是描述 抗病毒核苷（和天然的 存在的核苷）通过细胞内转运。 包括血脑屏障的细胞（内皮细胞）和 血-CSF屏障（脉络丛上皮细胞）。 这些 目标包括了解 转运蛋白，转运蛋白所在的膜位点，以及 底物和抑制剂的动力学迁移特性 的特异性 在这项提议中，大脑的核苷转运蛋白 内皮细胞和脉络丛将通过 互补克隆核苷转运缺陷型酵母（S. 酿酒酵母）转化的微血管和脉络丛cDNA 图书馆. 无钠和钠- 选择依赖性核苷转运蛋白并测序。 特异于所述多肽的推导的氨基酸序列的抗体 将准备转运蛋白并用于确定其细胞 位置和相对丰度利用光和超微结构 免疫细胞化学 钠依赖性核苷转运蛋白将 在杆状病毒表达系统中表达， 由电生理学确定的转运特性 技术. 这些研究的结果将有助于发展 a）优化抗HIV药物向大脑的递送，B） 确定潜在的抗病毒核苷对 通过快速体外方法设计核苷转运蛋白，和c）通过 新型抗病毒核苷转运体构效关系研究 有利于药物输送和抗病毒功效的性质。

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.