Bleomycin is a glycopeptide in regular clinical use to treat cancer. It is thought to attack tumor cells by carrying out metal, probably iron- dependent, DNA damage. It has two structural features, metal-binding and DNA-binding domains, which underlie the current understanding of its mechanism of action. The past decade has seen a major effort to define the mechanism of in vitro DNA damage. Still, many of the basic reactions of metallobleomycins which may be important for its interactions with DNA remain to be explored. It is our overall goal to understand the mechanism of inhibition of tumor cell proliferation caused by bleomycin in relation to its bioinorganic chemistry. At present the focus of our research is on the relationship of in vitro mechanisms of DNA strand breakage by bleomycin (Blm) to its ability to cause single and double strand cleavage at small ratios of drug to DNA base pairs in cells. To advance understanding in these areas, the following specific aims are set forth: 1) To define the 3-dimensional structures of selected metallobleomycins (M-Blm) in solution and bound to DNA. 2) To determine the kinetic properties of the association of MB1ms with DNA. 3) To explore the metal-binding properties of DNA-bound Blm. 4) To assess equilibrium, electronic and conformational aspects of adduct formation for metallobleomycins bound to DNA. 5) To specify kinetic and mechanistic features of DNA strand breakage at large base pair to FeBlm ratios. The experimental approaches to address these aims range from biochemical and chemical to biophysical in nature. A comprehensive examination of the chemistry of metallobleomycins, mainly iron bleomycin, bound to DNA will be undertaken using a variety of techniques that combine structural and mechanistic approaches to the definition of the role of metals in DNA strand scission. Two-dimensional pulsed NMR and ESR methods for structural analysis will be used to further our understanding of the 3-D structure of metallobleomycins in solution and bound to DNA. Various absorbance, fluorescence, and viscometric techniques will provide tools to follow the kinetics of reaction of drugs as they bind and react to DNA. These will be complemented by electrochemical methods to elucidate features of the redox chemistry of DNA-bound metallobleomycins that lead to DNA damage.
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