Nucleosides (e.g., adenosine) and synthetic nucleoside analogs (e.g., cytosine arabinoside (Ara-C) and 2',3'-dideoxyinosine (ddI)) are being used in the treatment of a wide array of disease states including cancer, viral infections, and cardiac arrhythmias. The overall goal of studies proposed in this competitive renewal application is to identify the structure, function and biological roles of Na+-dependent nucleoside transporters with particular emphasis on the specific transporters in the choroid plexus that are vital in the central nervous system (CNS) disposition and targeting of nucleosides and nucleoside drugs. In recent exciting studies, the first Na+-dependent nucleoside transporters were cloned from rat (rPUR and rPYR) and human (hPUR (cloned in this laboratory) and hPYR) tissues. hPYR and rPYR are pyrimidine-selective whereas hPUR and rPUR are purine- selective. Initial studies suggest that these cloned transporters are present in choroid plexus epithelium and play a role in the CNS targeting of nucleosides and nucleoside analogs. Novel studies performed in this laboratory with chimeric transporters have identified, for the first time, the gross structural domains responsible for the purine and pyrimidine selectivity of rPUR and rPYR.
The specific aims of the proposed studies are: (1) To determine the functional characteristics hPUR and hPYR in heterologous expression systems. Particularly, we will investigate the mechanisms of interactions of synthetic nucleoside analogs which are important in the treatment of cancer and viral infections; (2) To determine the functional domains and critical amino acids responsible for purine and pyrimidine substrate recognition in PUR and PYR; and (3) To localize rPUR and rPYR to the brush border or basolateral membrane of the choroid plexus. Briefly, we will perform functional studies in a mammalian expression system. Site-directed mutagenesis, construction of chimeric transporters together with molecular modeling will be used to determine the critical amino acids responsible for substrate selectivity in the transporters. Studies with antibodies will localize the transporters in choroid plexus epithelium. These studies will greatly advance our understanding of the molecular mechanisms involved in nucleoside transport and the structural elements of nucleoside transporters critical in purine and pyrimidine discrimination. The information gained is important in drug design and targeting to the CNS.
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