Bleomycin (Blm) is one of 3-4 highly effective drugs used to treat human cancer that require metals for their action. The iron complex of B1m(F3B1m) causes single and double strand scission of DNA in vivo and in vitro. The chemical properties of the redox activation of FeB1m to HO(2)(-)-Fe(III)B1m have been described in solution, but less is know about the reactions of FeB1m initiates DNA damage leading to inhibition of tumor growth. The central hypothesis is that DNA significantly influences the chemistry of FeB1m because the DNA domain and metal domain of the drug interact with the DNA polymer.
The specific aims are (1) To carry out structural analyses of metallobleomycin adducts bound to DNA sites, focusing on interaction of the metal domain with DNA and the mechanism by which drugs locate specific binding sites. (3) To determine properties of reduction of FeB1m species bound to DNA. (4) To investigate the mechanism of DNA damage including double strand cleavage of DNA by FeB1m. Using NMR, ESR, ENDOR and resonance raman techniques, structural features of relevant metallobleomycin species in solution and bound to DNA will be determined. These include CoB1mDNA structures to model important forms of FeB1mDNA and the deglycometal domain (Co, Fe) to examine the interaction of the metal domain with DNA. The electro-chemistry of FeB1mDNA will be examined using square wave voltammetry to define properties of redox states of FeB1m nd to provide unique means to generate HO(2)(-) -Fe(III)B1mDNA for studies of its reaction with DNA. Assays of DNA damage will assess the reaction of FeB1m with DNA in terms of kinetics of single and double strand cleavage. Site specific and nonspecific DNA oligomers will be synthesized and used in these studies. These experiments should markedly increase the understanding of how FeB1m, the prototype metallonuclease, achieves high efficiency of reaction with DNA.
Showing the most recent 10 out of 35 publications
