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Book of Abstracts - Ruhr-Universität Bochum

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P-46<br />

ISBOMC `10 5.7 – 9.7. 2010 <strong>Ruhr</strong>-<strong>Universität</strong> <strong>Bochum</strong><br />

New Topoisomerase II Poisons<br />

Matthew P. Akerman, a Mark T. Muller, b and Orde Q. Munro* a<br />

a University <strong>of</strong> KwaZulu-Natal, School <strong>of</strong> Chemistry, AuTEK Biomed, Private Bag X01, Scottsville,<br />

Pietermaritzburg, South Africa. b University <strong>of</strong> Central Florida, College <strong>of</strong> Medicine, Biomolecular<br />

Research Annex, 12722 Research Parkway, 32826-3227, Orlando, FL, USA.<br />

E-mail: munroo@ukzn.ac.za<br />

DNA topoisomerase II (topo II) is a well-established anticancer drug target. We have identified novel<br />

metallo-drugs that act specifically on topo IIA. Topo II enzymes are essential for life and are primarily<br />

responsible for decatenation <strong>of</strong> daughter chromatids during mitosis. 1 To function as a decatenase, topo<br />

II makes a transient double strand DNA break, providing an enzyme/DNA gate through which a distal<br />

duplex strand may pass. 1 The DNA cleavage intermediate is unique since a covalent DNA-topo II<br />

complex exists during the trans-esterification at the site <strong>of</strong> the break. Compounds that react with this<br />

transient intermediate, forming a ternary DNA-enzyme-drug complex, can arrest or poison the<br />

cleavage/religation cycle, inducing permanent DNA breaks, thereby damaging the genome <strong>of</strong> the<br />

target cell. Acute cytotoxicity results as the cell accumulates double strand DNA breaks. Drugs that<br />

induce breaks are topo II poisons and are generally excellent anti-cancer agents.<br />

We have synthesized and fully characterized a series <strong>of</strong> crystalline d 8 coordination compounds with<br />

tetradentate ligands. The compounds were screened by the National Cancer Institute (NCI, USA)<br />

against their panel <strong>of</strong> 60 human cancer cell lines. The most active compound is chiral, has a mean IC50<br />

<strong>of</strong> 14(2) �M, and is more cytotoxic than cisplatin (mean IC50 = 27 �M). Some cancer cell lines were<br />

substantially more susceptible to the new compounds than to cisplatin (ca. 12–30% <strong>of</strong> the cell lines<br />

tested, depending on the compound used). Statistical comparison <strong>of</strong> the ex vivo data for the most active<br />

compounds with drugs having known modes <strong>of</strong> action in the NCI database indicated that the cellular<br />

target is most likely topo II. This prediction was confirmed by in vitro DNA cleavage experiments<br />

using purified topo I and II and supercoiled DNA substrate. The data indicate that the compounds act<br />

as poisons at low concentrations (best current EC50 ∼ 1 �M) and as catalytic inhibitors at higher<br />

concentrations (typical EC50 ∼ 20–30 �M). The compounds are specific for topo II and do not target<br />

topo I, even at high concentrations. In vivo experiments are currently underway to assess whether the<br />

compound can target topo II in a chromatin setting. Preliminary data demonstrate that topo I is not<br />

being targeted in the cancer cell lines tested.<br />

Some <strong>of</strong> the compounds hydrolyze in aqueous buffer to generate metal-hydroxo derivatives. All<br />

hydrolysis-inert compounds bind calf thymus DNA (pH 7 phosphate buffer, 37 �C) with association<br />

constants ranging from 1.43(3) × 10 5 to 1.01(4) × 10 6 M –1 . The compounds with a high affinity for calf<br />

thymus DNA were all active cytotoxic agents in the NCI-60 screen. Reduction <strong>of</strong> the compounds by<br />

cellular levels <strong>of</strong> glutathione (pH 7 phosphate buffer, 37 �C) was followed by visible spectroscopy.<br />

Loss <strong>of</strong> the metal-to-ligand charge transfer (MLCT) band and appearance <strong>of</strong> the �–�* band <strong>of</strong> the free<br />

ligand confirmed reductive demetallation <strong>of</strong> the chelate in each case. The kinetics had second-order<br />

rate constants ranging from 0.0463(2) to 0.301(7) M –1 s –1 . Importantly, the most active compounds in<br />

the NCI-60 screen had the slowest reduction kinetics. Several structure–activity relationships for this<br />

new class <strong>of</strong> topoisomerase II poison have thus been delineated. A provisional patent has been filed<br />

and toxicology screens on the most active compounds have been scheduled.<br />

Acknowledgements: We thank AuTEK Biomed (Mintek and Harmony) for permission to publish selected data<br />

and financial support, the Department <strong>of</strong> Science and Technology (SA-COST EU Reciprocal Agreement) for a<br />

travel grant, and the Developmental Therapeutics Program (NCI, USA) cytotoxicity screens.<br />

References<br />

1. K. C. Dong, J. M. Berger, Nature 2007, 450, 1201-1205.<br />

104

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