The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg2+ self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg2+. Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg2+ complex and serve to define the position of this complex on the minor groove of DNA.
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