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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI033239-08
Application #
6169743
Study Section
Special Emphasis Panel (ZRG5-AAR (04))
Program Officer
Litterst, Charles L
Project Start
1992-04-01
Project End
2002-06-30
Budget Start
2000-07-01
Budget End
2002-06-30
Support Year
8
Fiscal Year
2000
Total Cost
$224,132
Indirect Cost
Name
University of Alabama Birmingham
Department
Pharmacology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
el Kouni, Mahmoud H (2002) Trends in the design of nucleoside analogues as anti-HIV drugs. Curr Pharm Des 8:581-93
Coghlan, M E; Sommadossi, J P; Jhala, N C et al. (2001) Symptomatic lactic acidosis in hospitalized antiretroviral-treated patients with human immunodeficiency virus infection: a report of 12 cases. Clin Infect Dis 33:1914-21
Kreimeyer, A; Schneider, B; Sarfati, R et al. (2001) NDP kinase reactivity towards 3TC nucleotides. Antiviral Res 50:147-56
Placidi, L; Cretton-Scott, E; Gosselin, G et al. (2000) Intracellular metabolism of beta-L-2',3'-dideoxyadenosine: relevance to its limited antiviral activity. Antimicrob Agents Chemother 44:853-8
Martin, L T; Cretton-Scott, E; Schinazi, R F et al. (1999) Pharmacokinetics of beta-L-2',3'-dideoxy-5-fluorocytidine in rhesus monkeys. Antimicrob Agents Chemother 43:920-4
Cui, L; Locatelli, L; Xie, M Y et al. (1997) Effect of nucleoside analogs on neurite regeneration and mitochondrial DNA synthesis in PC-12 cells. J Pharmacol Exp Ther 280:1228-34
Martin, L T; Faraj, A; Schinazi, R F et al. (1997) Effect of stereoisomerism on the cellular pharmacology of beta-enantiomers of cytidine analogs in Hep-G2 cells. Biochem Pharmacol 53:75-87
Fedorov, I I; Kazmina, E M; Gurskaya, G V et al. (1997) Novel 3'-C/N-substituted 2',3'-beta-D-dideoxynucleosides as potential chemotherapeutic agents. 1. Thymidine derivatives: synthesis, structure, and broad spectrum antiviral properties. J Med Chem 40:486-94
Fowler, D A; Rosenthal, G J; Sommadossi, J P (1996) Effect of recombinant human hemoglobin on human bone marrow progenitor cells: protection and reversal of 3'-azido-3'-deoxythymidine-induced toxicity. Toxicol Lett 85:55-62
Bridges, E G; Trentesaux, C; Lahlil, R et al. (1996) 3'-Azido-3'-deoxythymidine inhibits erythroid-specific transcription factors in human erythroid K562 leukemia cells. Eur J Haematol 56:62-7

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