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.
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