Protoberberines represent a new structural class of organic cations with selective cytotoxicity against CNS cancer cells. Their cytotoxic mechanisms have been demonstrated to involve poisoning of human DNA topoisomerases. Using purified topoisomerases, protoberberines are shown to poison both topoisomerases I and II (TOP1 and TOP2). These dual poisons of topoisomerases represent an emerging class of new anticancer agents with potential improved activity over either TOP I - or TOP2-specific poisons. The overall goal of this proposal is to understand the molecular mechanisms by which protoberberines differentially poison human TOP I and TOP2. Toward this goal, over one hundred protoberberines have been synthesized and evaluated for door ability to poison topoisomerases and kill a broad range of tumor cells. In addition to these biochemical and cell biological studies, we also have initiated biophysical studies of protoberberine interactions with DNA. Preliminary studies have suggested that a mixed mode of DNA binding (incorporating both minor groove-directed and intercalative interactions) is responsible for the dual poisoning activities of protoberberines. We hypothesize that the minor groove-directed interaction is critical for TOP I poisoning, while the intercalative interaction is important in TOP2 poisoning.
The Specific Aims of this proposal are: (a) To characterize and cross-correlate the DNA binding and topoisomerase poisoning properties of protoberberines. These studies are designed to provide evidence supporting the role of specific drug-DNA interactions (i.e. minor groove-directed versus intercalative) in differential topoisomerase poisoning. (b) To determine the role of minor groove-directed interactions in TOP I poisoning. Specifically, we will test the possibility that minor groove binding induces a DNA structural perturbation that results in stimulation of TOP1-mediated DNA cleavage. (c) To characterize specific drug-DNA and drug-enzyme interactions within the ternary cleavable complex using various biochemical methods, such as affinity labeling and protein modification interference. These studies are designed to assess the individual molecular interactions that govern the formation and stabilization of the ternary cleavable complex. Our proposed studies using this new class of topoisomerase poisons will enhance our understanding of the molecular basis for topoisomerase poisoning, which has been established as the key molecular event responsible for the antitumor activity of a broad -range of naturally occurring and synthetic compounds.
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