Self-Assembly of a Quinobenzoxazine-Mg2+ Complex on DNA: A New Paradigm for the Structure of a Drug-DNA Complex and Implications for the Structure of the Quinolone Bacterial Gyrase-DNA Complex

Abstract

The quinobenzoxazine compounds A-62176 and A-85226 belong to a novel class of antineoplastic agents that are catalytic inhibitors of topoisomerase II and also structural analogs of the antibacterial DNA gyrase inhibitor Norfloxacin. In vitro studies have shown that their antineoplastic activity is dependent upon the presence of divalent metal ions such as Mg2+ and Mn2+, although the precise role of these ions in the mechanism of action is unknown. In this study we have investigated the structures of the binary complex between the quinobenzoxazines and Mg2+ and the ternary complex between quinobenzoxazine-Mg2+ and DNA. The stoichiometry of the binary and ternary complexes and the biophysical studies suggest that a 2:2 drug:Mg2+ complex forms a "heterodimer complex" with respect to DNA in which one drug molecule is intercalated into DNA and the second drug molecule is externally bound, held to the first molecule by two Mg2+ bridges, which themselves are chelated to phosphates on DNA. There is a cooperativity in binding of the quinobenzoxazines to DNA, and a 4:4 drug:Mg2+ complex is proposed in which the two externally bound molecules from two different 2:2 dimers interact via pi-pi interactions. The externally bound quinobenzoxazine molecules can be replaced by the quinolone antibacterial compound Norfloxacin to form mixed-structure dimers on DNA. Based upon the proposed model for the 2:2 quinobenzoxazine:Mg2+ complex on DNA, a parallel model for the antibacterial quinolone-Mg2(+)-DNA gyrase complex is proposed that relies upon the ATP-fueled unwinding of DNA by gyrase downstream of the cleavable complex site. These models, which have analogies to leucine zippers, represent a new paradigm for the structure of drug-DNA complexes. In addition, these models have important implications for the design of new gyrase and topoisomerase II inhibitors, in that optimization for structure-activity relationships should be carried out on two different quinolone molecules rather than a single molecule.