BLOOD-BRAIN TRANSPORTERS OF ANTIVIRAL NUCLEOSIDES

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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.