The objectives of this competing renewal application are to continue and extend ongoing studies on the mechanism(s) by which pyrimidine nucleoside analogs active against the human immunodeficiency virus (HIV), such as 3'azido-3'- deoxythymidine and congeners, exert toxicity on bone marrow cells and other toxicity sites. The elucidation of these mechanism(s) may permit the development of a combination therapy with modulating agents that protect or reverse host toxicity without impairment of anti-HIV activity of the drug under scrutiny. This project includes two major aims: (1) the description of the biochemical and molecular mechanism(s) responsible for the effects of AZT and its metabolite, 3'-amino-3'- deoxythymidine (AMT) in human bone marrow cells; and (2) the detailed characterization of the enzymatic reduction of AZT to AMT in human hepatocytes with the evaluation of potential drug-drug interactions through the cytochrome P450 pathway. The Principal Investigator and his associates will define the mechanism(s) by which these 2',3'- dideoxynucleosides (ddN's) affect directly uridine-5'-monophosphate (UMP) synthase and indirectly other enzymes in the pathway for de novo pyrimidine biosynthesis. They will delineate the effects of these ddN's using human bone marrow liquid cultures in suspension and cells deficient in synthase (orotic aciduria cells) and with transformed cells with amplified enzyme activity; they will examine the potential alterations in the UMP synthase protein with discrimination of its two activities (i.e., orotate phosphoribosyltransferase [OPRT] and orotidine-5'-monophosphate decarboxylsse [OFC]). The analysis of mRNA steady-state levels of either gene affected by AZT and other analogs with the evaluation of the functionality of the RNA molecules transcribed by the affected gene will permit the determination of critical factors potentially important for effects of AZT on UMP synthase. Using human cultured muscle cells the researchers will ascertain whether inhibition of UMP synthase, as compared to the effects on mitochondrial DNA synthesis, plays a role in AZT-induced myopathies. With regard to the second aim, the researchers will use specific monoclonal antibodies and specific inhibitors to elucidate the cytochrome P450 isoenzyme responsible for conversion of 3'-azido-ddN's to 3'-amino-ddN's together with the role of NADPH P450 reductase in that reaction. They will assess the presence of this metabolism at target sites (lymphocytes) and toxic sites (bone marrow and muscle cells) with possible formation of a reactive intermediate an/or free radicals, and will determine the effects of cytochrome P450 inducers or inhibitors on AMT formation in human hepatocytes to ascertain whether AZT pharmacodynamic properties may be altered under these conditions.
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