The long term objectives of this renewal application are to investigate and extend ongoing studies on the cellular and molecular mechanism(s) by which nucleoside analogs selectively target the reverse transcriptase of the human immunodeficiency virus (HIV). Extensive studies on the interaction of these drugs with the host cell have been shown in the past funding period to be critical in our understanding of the underlying mechanism(s) of drug action and toxicity. The synthesis of novel L-purine nucleoside analogs and the PI's recent discovery that a new series of L-dideoxyadenosine analogs are potent inhibitors of HIV and HBV replication demonstrates for the first time that development of L-purine derivative should also be vigorously pursued. These data provide a strong rationale for elucidation of their mechanisms of action and metabolism in target cells of these viral infections. This project includes three major aims: (I) Identification of the metabolic pathways and mechanism of action of L-purine analogs with particular emphasis on L-dideoxyadenosine derivatives. This proposal will evaluate the cellular metabolism (anabolism and catabolism) and compartmentalization of L-ddA, L-d4A and derivative in established cell lines and in human primary cells which are infectable by HIV, including lymphocytes and monocyte-macrophages. Selective inhibitors and mutant genetically defective (dCK-, AK-, etc.) cells will be used to elucidate activating kinase enzymes involved in the activation processes of these nucleosides. Nucleoside kinases responsible for nucleoside anabolism will be purified and kinetic constants for the various nucleosides will be determined. Effect of structural alterations on substrate specificities targeted toward maximization of antiviral activity will be assessed. The impact of stereoisomerism of the beta-enantomers of purine analogs on their cellular metabolism as well as their interaction with the antiviral target and/or hypothesized toxicity sites will be examined, including the assessment of potential combination therapies through metabolic interactions. (II) Evaluation of rationally designed pronucleotides incorporating enzyme-mediated bioreversible protection groups allowing the direct intracellular delivery of b-L-nucleoside-5'-monophosphate. (III) Investigations of the potential for HIV-1 resistance development to L-purine nucleoside analogs by phenotypic and genotypic characterization; confirmation of the role of specific mutations by site-specific mutational analysis; examination of potential cross-resistance to clinically approved nucleoside analogs; impact of L-purine nucleoside resistant mutation of HIV-RT on substrate/inhibitor recognition by beta-L-5'-triphosphate derivatives.
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