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PLoS One. 2014 May 8;9(5):e97008. doi: 10.1371/journal.pone.0097008. eCollection 2014.

Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.

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  • 1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
  • 2Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America.


Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α-mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

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