Structural and solution determinants of DNA intercalation and topoisomerase IB inhibition by flavonoids

The DNA maintenance enzyme topoisomerase IB is thought to play crucial roles in living cells, and for this reason, inhibitors of this enzyme have been extensively studied. A gel electrophoresis method capable of characterizing and quantifying inhibition of topoisomerase IB by small molecular weight compounds was developed. The method uses closed plasmid DNA, and can separate single-strand nicked, linearized, fully relaxed, supercoiled forms and topoisomers of the plasmid, so that topoisomerase-dependent DNA cleavage can also be determined. Inhibitors of topo I are associated with intercalative binding to DNA, and the method can also simultaneously determine intercalative binding, except in the cases where topoisomerase inhibition is prohibitively strong. By quantifying poisoning, inhibition and intercalation simultaneously and separately in relation to reference compounds, it is possible to make quantitative determinations for comparative purposes. Data is presented for the topo I inhibitor luteolin. An additional 33 polyphenolic compounds, primarily flavonoid glycones and aglycones, were assayed for their ability to inhibit topo I and to intercalate DNA. It is shown that the most potent topo I poisons are the flavones and flavonols and that these generally, but not always, exhibit measurable DNA intercalation. There was no clear correlation, however, of topo I poisoning activity with degree of DNA unwinding. Surprisingly, both DNA intercalation and topo I poisoning was shown to occur with some flavon(ol) glycones, including the C-glycosylflavone orientin. Inhibition of relaxation by flavonoids was found to be difficult to quantify and most likely due to non-specific inhibition through flavonoid self-aggregation. An investigation of the acid-base chemistry of flavonoids determined that many flavonoids show acid-base activity with a pKa in the physiological pH region. For this reason, subtle pH changes may significantly affect the solution activity of flavonoids and their concomitant biological activity. In addition, these effects may be complicated by pH-dependent aggregation and oxidative degradation. Finally, a simple model for the intercalation of flavonoids into DNA was developed. A discussion of the possible consequences of intercalation and topoisomerase inhibition on a number of cellular processes, particularly with regard to the very low intracellular concentrations attained by flavonoids, follows.