Book of Abstracts - Ruhr-Universität Bochum

P-46
ISBOMC `10 5.7 – 9.7. 2010 Ruhr-Universität Bochum
New Topoisomerase II Poisons
Matthew P. Akerman, a Mark T. Muller, b and Orde Q. Munro* a
a University of KwaZulu-Natal, School of Chemistry, AuTEK Biomed, Private Bag X01, Scottsville,
Pietermaritzburg, South Africa. b University of Central Florida, College of Medicine, Biomolecular
Research Annex, 12722 Research Parkway, 32826-3227, Orlando, FL, USA.
E-mail: munroo@ukzn.ac.za
DNA topoisomerase II (topo II) is a well-established anticancer drug target. We have identified novel
metallo-drugs that act specifically on topo IIA. Topo II enzymes are essential for life and are primarily
responsible for decatenation of daughter chromatids during mitosis. 1 To function as a decatenase, topo
II makes a transient double strand DNA break, providing an enzyme/DNA gate through which a distal
duplex strand may pass. 1 The DNA cleavage intermediate is unique since a covalent DNA-topo II
complex exists during the trans-esterification at the site of the break. Compounds that react with this
transient intermediate, forming a ternary DNA-enzyme-drug complex, can arrest or poison the
cleavage/religation cycle, inducing permanent DNA breaks, thereby damaging the genome of the
target cell. Acute cytotoxicity results as the cell accumulates double strand DNA breaks. Drugs that
induce breaks are topo II poisons and are generally excellent anti-cancer agents.
We have synthesized and fully characterized a series of crystalline d 8 coordination compounds with
tetradentate ligands. The compounds were screened by the National Cancer Institute (NCI, USA)
against their panel of 60 human cancer cell lines. The most active compound is chiral, has a mean IC50
of 14(2) �M, and is more cytotoxic than cisplatin (mean IC50 = 27 �M). Some cancer cell lines were
substantially more susceptible to the new compounds than to cisplatin (ca. 12–30% of the cell lines
tested, depending on the compound used). Statistical comparison of the ex vivo data for the most active
compounds with drugs having known modes of action in the NCI database indicated that the cellular
target is most likely topo II. This prediction was confirmed by in vitro DNA cleavage experiments
using purified topo I and II and supercoiled DNA substrate. The data indicate that the compounds act
as poisons at low concentrations (best current EC50 ∼ 1 �M) and as catalytic inhibitors at higher
concentrations (typical EC50 ∼ 20–30 �M). The compounds are specific for topo II and do not target
topo I, even at high concentrations. In vivo experiments are currently underway to assess whether the
compound can target topo II in a chromatin setting. Preliminary data demonstrate that topo I is not
being targeted in the cancer cell lines tested.
Some of the compounds hydrolyze in aqueous buffer to generate metal-hydroxo derivatives. All
hydrolysis-inert compounds bind calf thymus DNA (pH 7 phosphate buffer, 37 �C) with association
constants ranging from 1.43(3) × 10 5 to 1.01(4) × 10 6 M –1 . The compounds with a high affinity for calf
thymus DNA were all active cytotoxic agents in the NCI-60 screen. Reduction of the compounds by
cellular levels of glutathione (pH 7 phosphate buffer, 37 �C) was followed by visible spectroscopy.
Loss of the metal-to-ligand charge transfer (MLCT) band and appearance of the �–�* band of the free
ligand confirmed reductive demetallation of the chelate in each case. The kinetics had second-order
rate constants ranging from 0.0463(2) to 0.301(7) M –1 s –1 . Importantly, the most active compounds in
the NCI-60 screen had the slowest reduction kinetics. Several structure–activity relationships for this
new class of topoisomerase II poison have thus been delineated. A provisional patent has been filed
and toxicology screens on the most active compounds have been scheduled.
Acknowledgements: We thank AuTEK Biomed (Mintek and Harmony) for permission to publish selected data
and financial support, the Department of Science and Technology (SA-COST EU Reciprocal Agreement) for a
travel grant, and the Developmental Therapeutics Program (NCI, USA) cytotoxicity screens.
References
1. K. C. Dong, J. M. Berger, Nature 2007, 450, 1201-1205.
104