Current therapies for AIDS have been directed at the viral reverse transcriptase (RT). 2',3'-Dideoxynucleosides (ddN), analogs of endogenous deoxynucleosides (dNs), following sequential phosphorylation to 5'-triphosphate (ddNTP), inhibit RT. Since ddNs metabolism is cell- species dependent, the kinetics of their metabolism and the cellular ddNTP pools will differ among target cells and be indicative of sensitivity and resistance to the drug. Long term objective of present investigation is to understand intracellular differences of ddN metabolism, their interactions with endogenous nucleotide pool and mechanisms of resistance in human interactions with endogenous nucleotide pool and mechanisms of resistance in human target cells and exploit this knowledge to selectively optimize their anti-AIDS efficacies. Therefore, the specific aims of this proposal are: (1) to compare ddN metabolism in human monocytic/macrophage cell line (U937), lymphocytic cell lines (CEM) and macrophages; (2) to determine their effects on endogenous nucleotide pool; (3) to determine how the nucleotide pool and the ddN metabolism differ in resistant cells; and (4) to determine the effects of modulators of ddN metabolism on ddNTP and endogenous nucleotide pool. Using human cells of different lineage and stages of differentiation which are permissive to HIV and the potential dideoxynucleosides currently available (dideoxynucleosides of adenine, hypoxanthine, cytosine, guanine and azido-dideoxythymidine), and the new drugs available through our drug development program, the following studies will be carried out: activities and the kinetic paramenters of the key anabolic and catabolic enzymes using ddNs as substrates; intracellular accumulation and degradation of dideoxynucleotides; interactions of ddNs on the existing endogenous nucleotide pool, and their de novo and salvage synthetic pathways will be determined by incorporating radiolabled purine and pyrimidine nucleosides and their precursors (glycine and NaHCO3) in the nucleotide pools. To determine the mechanisms of resistance, the above studies will be performed in cells which had been continuously exposed to the ddNs. Effects of a novel class of compound (nucleotide dimers) which may provide cells with the prephosphorylated two active drugs; inhibitors of ddI and ddG catabolism: and intervention at de novo and salvage pathway of purine pyrimidine nucleotides synthesis to reduce levels of dNTP will also be examined.
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