title: Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.
abstract: 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.
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Yu-Shih
authors.initials: YS
authors.lastname: Lin
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Wan-Chen
authors.initials: WC
authors.lastname: Huang
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Mei-Shya
authors.initials: MS
authors.lastname: Chen
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Tao-Shih
authors.initials: TS
authors.lastname: Hsieh
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004347:Drug Interactions; D004354:Drug Screening Assays, Antitumor; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008958:Models, Molecular; D058990:Molecular Targeted Therapy; D009690:Nucleic Acid Conformation; D010472:Perchlorates / Q000494:pharmacology; D017670:Sodium Compounds / Q000494:pharmacology; D013713:Teniposide / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology*
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D010472:Perchlorates; D017670:Sodium Compounds; D059005:Topoisomerase II Inhibitors; D004247:DNA; D013713:Teniposide; C031068:sodium perchlorate; D004250:DNA Topoisomerases, Type II