The overall aim of this proposal is to study the biochemical pharmacology of anti-human immunodeficiency virus type (HIV-1) compounds. The current compounds of interest will be anti-HIV nucleoside analogs with a special focus on 4'-ethynyl D4T (4'-Ed4T), which was discovered in this laboratory in collaboration with two Japanese groups. This compound has unique anti-HIV activity different from other anti-HIV nucleoside analogs. We propose to study the biochemical pharmacology of this newly discovered anti-HIV D-thymidine analog, 4'-Ed4T.The following specific aims are based on these original findings in my laboratory. 1. Metabolism of 4'-Ed4T in cells. a. Study the time dependent phosphorylation of 4'-Ed4T. b. Study the dose dependent formation of 4'-Ed4T nucleotides. c. Study the intracellular concentration of 4'-Ed4T nucleotide metabolites after drug removal. d. Study the cellular enzymes responsible for the phosphorylation of 4'-Ed4T in cells. 2. Interaction of 4'-Ed4T metabolites with HIV reverse transcriptase. 3. Impact of 4'-Ed4T host on DMA synthesis. 4. Characterization of HIV resistant to 4'-Ed4T. a. Engineering 4'-Ed4T resistant mutants and sensitivity of the defined mutation virus to 4'-Ed4T. b. To determine the replication capacity (RC) coefficient (calculations described below) of 4'-Ed4T resistant strains. c. To understand the impact of mutations P119S, T165A and M184V on HIV RT behavior. The studies proposed will provide not only the key preclinical information about the newly discovered 4'- Ed4T which will be under clinical development and the structure activity relationship of the biochemical determinants studied, but also the role of potential biochemical determinants for the action of thymidine analogs including AZT and D4T. 4'-Ed4T could have the potential to replace AZT or D4T in anti-HIV HARRT and in overcoming drug resistant virus to other anti-HIV drugs at the relevant dosage without causing toxicity. ? ? ? ? ? ?
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