The inhibition of nucleic acid metabolism has long been recognized as a powerful chemotherapeutic target having one significant pitfall - non-selective killing of both diseased and healthy cells. We propose a unique strategy to potentiate the effects of existing chemotherapeutic agents by using nucleoside analogs that display preferential insertion opposite damaged DNA versus their unmodified counterparts. By targeting promutagenic DNA synthesis, these analogs will hinder the development of drug resistance and other diseases caused by inappropriate replication of damaged DNA.
Aim 1 will use our experience in medicinal chemistry and enzymology to further develop a series of novel nucleoside analogs that are preferentially inserted opposite this DNA lesion. We shall employ a kinetic approach to generate a structure/function relationship for each non-natural analog to correlate the catalytic efficiency of insertion with the unique electronic signature of the substituted analog. Completion of this Aim will provide evidence for rationally targeting DNA lesions, the ability to selectively inhibit DNA polymerases, and define the contributions of p-electron interactions in DNA polymerization. We hypothesize that these non-natural nucleosides will selectively inhibit polymerases involved in DNA repair and/or error-prone synthesis. They are therefore predicted to potentiate the effects of chemotherapeutic agents that induce DNA damage.
Aim 2 will test the functionality of these analogs by quantitatively evaluating the efficacy of each analog at the cellular level. The anti-proliferative effects of our nucleoside analogs will be addressed through a series of cell cytotoxicity studies using a T-lymphoblast cancer cell line (CEM-7) as our initial model. After defining the potency and selectivity of each non-natural nucleoside analog, we shall characterize the cellular pathways accounting for the induction of cell death and/or anti-proliferative behavior to describe their mechanism of action. Finally, the potentiating effects of these analogs will be evaluated by measuring the cytotoxic effect of the nucleosides used in conjunction with DNA damaging agents. Completion of this Aim will provide a new paradigm for treatment. Cancer is frequently characterized by cell cycle deregulation involving altered activity of cyclin-dependent kinases.
Aim 3 will test the ability of the non-natural analogs to inhibit cell-cycle progression regulated by this class of kinase. In vitro experiments will delineate the mechanism of action. Results generated from this Aim will provide a greater understanding of the enzymes involved in regulating the cell cycle. This knowledge will facilitate the development of new drugs as the repertoire of targets becomes more differentiated.
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