Topoisomerase II Binds to Ellipticine in the Absence or Presence of DNA.

Abstract

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

Topoisomerase Poisons: Harnessing the Dark Side of Enzyme Mechanism

“…Support for this mechanism comes from recent spectroscopy studies on the interaction of intoplicine or ellipticine with topoisomerase II (54,72). In both cases, drugs complexed with the enzyme in the absence of DNA.…”

“…In contrast, agents targeted to topoisomerase II have been shown to utilize both mechanisms. While etoposide and amsacrine are potent inhibitors of religation (14,53), agents such as quinolones and ellipticine have little effect on this enzyme activity and presumably act by enhancing the forward rate of cleavage (14,35,54). Comparable studies have not been carried out for DNA gyrase.…”

“…Many of these enzymes still maintain high catalytic activity, suggesting that changes in sensitivity result from altered protein⅐drug interactions. Second, a recent fluorescence spectroscopy study on topoisomerase II⅐ellipticine interactions (54) demonstrated that the enzyme dictates the protonation state of the drug in the ternary complex. Moreover, the fluorescence lifetime of bound ellipticine was similar in the binary (enzyme⅐drug) and ternary complexes, indicating a similar drug environment in both.…”

Spontaneous DNA Damage Stimulates Topoisomerase II-mediated DNA Cleavage

“…For example, anticancer drugs such as etoposide (see Fig. 5, inset) and amsacrine enhance topoisomerase II-mediated DNA scission primarily by inhibiting DNA religation (48 -50), while other agents such as quinolones and ellipticine act primarily by increasing the rate of cleavage complex formation (51,52). Finally, cleavage-enhancing apurinic sites have essentially no effect on rates of DNA religation and presumably act by stimulating the rate of cleavage complex formation (27).…”

The origin of the dual fluorescence of protonated ellipticine in water

“…The markedly different fluorescence features of E and EH + enabled the detection of the extent of protonation in different intracellular compartments [7] and the proton movement in mitochondria [8]. Time-resolved fluorescence measurements showed the substantial variation of the fluorescence lifetime upon binding to various subcellular constituents [9].…”