BINDING OF METALLOBLEOMYCINS TO DNA 10 MERS

Medical College Of Wisconsin, Milwaukee WI

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Abstract

Iron bleomycin (FeBlm) is an anticancer metallodrug, containing metal and DNA binding domains within its structure, which causes DNA strand breakage and base release through a redox mechanism. According to an x-ray crystallographic study of a metal domain fragment of CuBlm and an NMR analysis of peroxy-CoBlm, the metal exists in a square planar environment of five nitrogen donor atoms. When HO2-Co(III)Blm binds to DNA oligomers containing 5'-GC-3' or 5'-GT-3' recognition sites, it binds specifically to these sites through both its metal and DNA binding domains. In the present study, the binding of several metallobleomycins (MBlm) to d(GGAAGCTTCC)2 was examined by EPR, CD, and fluorescence spectroscopies. EPR spectra for Co(II)Blm in the absence or presence of 10-mer were resolved in the g-parallel region into a five-line superhyperfine pattern consistent with the coordination of two axial nitrogen ligands from the primary amine and an amide of the structure. Addition of Blm and MBlm [M=Zn, Cu, Fe(III), HO2-Co(III)] to 10-mer caused significant perturbation of the CD spectrum of bound DNA in only the last two MBlm species, indicating that only their metal domains interacted strongly with the DNA structure. The binding constants of each of these drug-DNA adducts were determined with fluorescence titrations at 25C and pH 7.4 as 1.6 x 10(4), 8x10(4), 8x10(4), 3x10(5), and 4x10(6), respectively. Considering that charges on the metal domains of these forms show no regular variation in relation to binding constant, it is concluded that differences in the coordination environment and their chirality contribute to these sizable differences. Such results will be compared with others based on the binding of MBlms to d(GGAAATTTCC)2, which does not contain a specific site of drug binding.[unreadable]