| Summary: | 博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 101 === Type II topoisomerases (Top2s) are essential enzymes responsible for the timely resolution of DNA topological problems by temporally cleaving both strands of a DNA duplex to allow the passage of another. Agents that perturb the Top2-mediated DNA cleavage/rejoining process consist of a group of successful clinically active anticancer drugs, including etoposide, doxorubicin, amsacrine (m-AMSA) and mitoxantrone. Bothe isoforms of human Top2, 2α and 2β, can be targeted by these agents, in which the enzymes are trapped on DNA in the form of covalent enzyme-DNA adduct, termed Top2 cleavage complex (Top2-cc). The drug-induced accumulation of Top2-cc on DNA leads to fragmentation of genomic DNA and cell death. Despite their potent anticancer activities, a wider application of Top2-targeting drugs is hampered by deleterious side effects and the emergence of drug-resistant cells. Among them, the therapy-related leukemia is a thorny side effect particularly raised by Top2-based chemotherapy, and is considered induced by Top2β-targeting of drugs, which introduces double-strand break on regulatory region of genes and leads to genome rearrangement. This calls for an isoform-specific Top2-targeting strategy that may suppress such life-threatening side effect of this sort of drugs. To facilitate development of next generation Top2-targeting agents, it is essential to understand the structural basis of drug action in detail. Therefore, in the present study, the crystal structure of an etoposide-bound human Top2β cleavage complex was determined, which reveals a DNA cleavage site-specific drug insertion and a concomitant decoupling of active site residues, thus explaining how the drug blocks the rejoining of broken DNA ends. In addition, we established a post-crystallization drug replacement procedure to simplify the structural analysis of other Top2-targeting drugs, by which the structures of m-AMSA-, doxorubicin- and mitoxantrone-stabilized hTop2β cleavage complexes were successfully determined. While m-AMSA and mitoxantrone also targeted to DNA cleavage sites as expected, however, doxorubicin bound at an unusual location aside from the typical drug-binding pocket. For those structures derived from drug-replacing procedure, the structures bound by m-AMSA and mitoxantorne nicely explain reported drug-resistant mutation and structural-activity relationships of the two drugs. In contrast, the binding mode of doxorubicin is not consistent with the known properties, but nevertheless implies doxorubicin may adopt a unique inhibiting mechanism different from other Top2-targeting agents. By recognizing the conformational landscapes of the drug-binding pockets and those drug-interacting residues that are different between human Top2α and 2β, we propose the guidelines for design of isoform-specific Top2-targeting agents.
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