Phenanthriplatin Acts As a Covalent Poison of Topoisomerase II

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Phenanthriplatin Acts as a Covalent Poison of Topoisomerase II Cleavage Complexes Imogen A. Riddell, Keli Agama, Ga Young Park, Yves Pommier, and Stephen J. Lippard ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00565 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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ACS Chemical Biology

Phenanthriplatin Acts as a Covalent Poison of Topoisomerase II Cleavage Complexes Imogen A. Riddell,a Keli Agama,b Ga Young Park,a Yves Pommier,b,† and Stephen J. Lipparda,† a

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 b Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892

Abstract Drugs capable of trapping topoisomerase II (Top2), an essential enzyme that cleaves DNA to remove naturally occurring knots and tangles, can serve as potent anticancer agents.

The

monofunctional

platinum

agent

phenanthriplatin,

cis-

[Pt(NH3)2(phenanthridine)Cl](NO3), is shown here to trap Top2 in addition to its known modes of inhibition of DNA and RNA polymerases. Its potency therefore combines diverse modes of action by which phenanthriplatin kills cancer cells. The observation that phenanthriplatin can act as a Top2 poison highlights opportunities to design non-classical platinum anticancer agents with this novel mechanism of action. Such complexes have the potential to overcome current limitations with chemotherapy, such as resistance, and to provide treatment options for cancers currently that do not respond well to classical agents. Covalent DNA-platinum lesions implicated in Top2 poisoning are distinctive from those generated by known therapeutic topoisomerase poisons, which typically exert their action by reversible binding at the interface of Top2-DNA cleavage complexes.

Introduction Nuclear topoisomerases are essential for replication, transcription, repair, and chromosome organization. Specifically, topoisomerases resolve topological problems inherent to the double-helical structure of DNA, unwind supercoiled DNA and untangle inter- and intra-molecular DNA knots and catenanes. Type 1 and type 2 topoisomerases, which include topoisomerase I (Top1) and topoisomerase II (Top2),1-6 respectively, are

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2 distinguished by their differential catalytic intermediates. Top1 enzymes cleave a singlestrand of DNA to which they bind, whereas Top2 enzymes cleave both strands of the DNA substrate.1-6 In vertebrates, Top2 exists as two closely related isoforms Top2α and Top2β.7,8 These two isoforms have greater than 70% amino acid similarity but are differentially regulated within the cell.4,6,9 Top2α is essential for cell growth and is expressed in high levels in rapidly proliferating and cancer cells; its expression peaks at the G2/M phase of the cell cycle. By contrast, the concentration of the β isoform is more or less constant throughout the cell cycle and coupled with transcription.6,10-13 The two isoforms share a common mechanism of action.14 Following DNA binding of each enzyme, a DNA cleavage/religation equilibrium is established. In this step a double-strand break is generated from two nicks on opposite sides of the DNA separated by a four base pair 5’ overhang. Transesterification initiated by nucleophilic attack of a tyrosine residue of Top2 generates a covalent phosphotyrosyl bond and the resultant covalently bound DNA:Top2 complex is referred to as the cleavage complex: Top2cc.15 Following formation of Top2cc, binding of an ATP cofactor facilitates reconfiguration of the enzyme and DNA strand passage. Religation regenerates doublestranded DNA differing from that of the starting DNA only by its topological properties. Finally, hydrolysis of an enzyme-bound ATP molecule triggers release of the untangled DNA.14 Although this enzymatic action is essential to life, it is also an important target in cancer treatment.9,16,17 Under normal circumstances Top2cc is a transient intermediate, present at low steady state levels in the catalytic cycle, and the cleavage/religation equilibrium in which it is established is readily reversible, lying in favor of the ligation reaction.5,6 Top2 poisons exert their mode of action by binding to the Top2cc to generate a ternary complex16,18,19 that may either prevent religation or promote the forward cleavage reaction.20 The cytotoxic effects of this class of compounds do not result from a suppression of the catalytic activity of Top2, but instead, from the stalling of Top2cc, which blocks polymerases and interferes with chromatin structure and genomic stability, resulting in DNA fragmentation and ultimately cell death.15-17


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3 The early discovery that the Top2 catalytic cycle could be exploited in the development of anticancer drugs has resulted in significant research in this field, both in elucidating the mechanism of action of different Top2 poisons21,22 and inhibitors23 and in designing novel complexes with better anticancer properties and reduced side effects.15,20,24 Topoisomerase poisons and inhibitors are used extensively in the clinic, with almost every form of cancer sensitive to chemotherapy being treated, at least partially, with drugs that target topoisomerases. The majority of Top2 drugs currently in the clinic do not distinguish between the two Top2 isoforms, but research indicates that the α form is the primary cytotoxic target whereas drugs that target the β form have toxic side effects and are linked to the development of secondary malignances.12,15,25-27 Phenanthriplatin is a promising anticancer agent despite violating classical structure activity relationships (SAR’s) proposed for platinum anticancer complexes.28 Previously we reported that this cationic platinum(II) complex exerts its mode of action through binding to DNA in a monofunctional fashion, binding to only a single nucleotide rather than forming a cross-link like cisplatin, blocking the procession of both RNA29,30 and DNA31 polymerases along the DNA. In the present study we describe the results of experiments designed to test the hypothesis that a significant contribution to the potency of phenanthriplatin may be its ability to act as a Top2 poison. Analysis of phenanthriplatin using the COMPARE algorithm of the NCI60 screening panel32 and CellMiner33 provided the initial indication that the monofunctional agent might be acting as a Top2 inhibitor.28 A representative subset of the results is shown in Table 1. The data indicate that the effects of phenanthriplatin correlate well with those of known Top2 poisons, while differing from those of tubulin inhibitors and other N7-alkylating agents, including the commonly used platinum agents cisplatin, carboplatin, and oxaliplatin. The full data set, containing the top 2,000 of 20,000 compounds obtained using the CellMiner Pattern Comparison tool,33 is supplied as a supplemental data file.



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4 Table 1: Correlation between phenanthriplatin and Top2 inhibitors (T2, red), N7alkylating agents (A7, blue), and tubulin inhibitors (Tu, green) determined using the COMPARE algorithm of the NCI60 screening panel.33 Significantly higher correlations were observed between the Top2 inhibitors than with the other platinum agents. NSC 759703 123127 301739 349174 82151 164011 337766 246131 249992 122819 256439 141540 707389 178248

Name Phenanthriplatin Doxorubicin Mitoxantrone Oxanthrazole Daunorubicin Rubidazone Bisantrene hydrochloride Valrubicin M-AMSA Teniposide Idarubicin Etoposide Eribulin mesilate/mesylate Chlorozotocin

MOA T2 T2 T2 T2 T2 T2 T2 T2 T2 T2 T2 T2 A7

Correlation 1 0.519 0.517 0.494 0.446 0.438 0.433 0.381 0.358 0.336 0.297 0.286 0.282 0.254

95466 750 135758 9706 353451 338947 182986 119875 6396 241240 25154 762 34462 3088 8806 138783 67574 77213 363812 409962 628503 109724 266046 271674 85998 45388 26271

PCNU Busulfan Piperazinedione Triethylenemelamine Mitozolomide Clomesone AZQ Cisplatin Thiotepa Carboplatin Pipobroman Mechlorethamine Uracil mustard Chlorambucil Melphalan Bendamustine Vincristine sulfate Procarbazine hydrochloride Tetraplatin Carmustine Docetaxel,Docetaxol Ifosfamide Oxaliplatin Carboxyphthalatoplatinum Streptozocin Dacarbazine Cyclophosphamide

A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 A7 Tu A7 A7 A7 Tu A7 A7 A7 A7 A7 A7

0.232 0.205 0.203 0.202 0.201 0.199 0.195 0.185 0.178 0.175 0.172 0.143 0.14 0.138 0.108 0.097 0.095 0.093 0.046 0.046 0.041 0.013 0.006 -0.013 -0.112 -0.301 -0.382

Two independent methods were employed to test the formation of Top2cc in cancer cells. Both analyses indicated that treatment of HT-29 human colorectal adenocarcinoma cells with phenanthriplatin at increasing time intervals resulted in an


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5 increase in Top2cc, the transient, covalent DNA-Top2 complex normally observed at low levels in cells. First, an in-vivo complex of enzyme (ICE) assay34,35 (Figure 1A-B) indicated that, after treatment with phenanthriplatin (50 µM for 1 h), HT-29 cell extracts contained more Top2cc than was observed in a control reaction that lacked a Top2 poison. Quantitation of independent experiments showed that phenanthriplatin induced a significant amount of Top2cc at 3 and 6 h exposure. In these experiments, etoposide, a well-established Top2 poison,9,16 was employed as a positive control and showed the efficiency of phenanthriplatin at inducing Top2cc (Figure 1B).

Figure 1: Induction of Top2 cleavage complexes (Top2cc) by phenanthriplatin in human colon cancer HT-29 cells. A) Representative blot taken from cells treated with 100 µM etoposide for 3 h, or 50 µM phenanthriplatin (Phen-Pt) for 1, 2, 3 or 5 h. Equal numbers of cells were lysed in DNAzol reagent and submitted to the ICE assay. 34,35 Different concentrations of genomic DNA (0.2, 0.1, 0.05 and 0.025 µg) were probed with an antiTop2 antibody. Etoposide was used as a positive control for Top2cc formation. B) Quantitation of Top2cc measured by the ICE assays in four independent experiments. C) Cells treated with phenanthriplatin for 5 h were detected by the alkaline elution assay. 36 Quantification of DNA–Top2 cross-linking (in rad equivalents [rad-eq]). Data at each concentration of phenanthriplatin represent three independent determinations. * p

Phenanthriplatin Acts As a Covalent Poison of Topoisomerase II

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