Structural and Metal Ion Effects on Human Topoisomerase IIα

Article Cite This: Chem. Res. Toxicol. 2019, 32, 90−99

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Structural and Metal Ion Effects on Human Topoisomerase IIα Inhibition by α‑(N)-Heterocyclic Thiosemicarbazones William H. Morris,† Lana Ngo,† James T. Wilson,§ Wathsala Medawala,‡ Anthony R. Brown,† Jennifer D. Conner,† Florence Fabunmi,† Derek J. Cashman,† Edward C. Lisic,† Tao Yu,† Joseph E. Deweese,*,§,∥ and Xiaohua Jiang*,† †

Department Department § Department 37204-3951, ∥ Department

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‡

of Chemistry, Tennessee Technological University, Cookeville, Tennessee 38505, United States of Chemistry, Georgia College, Milledgeville, Georgia 31061, United States of Pharmaceutical Sciences, Lipscomb University College of Pharmacy and Health Sciences, Nashville, Tennessee United States of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States

S Supporting Information *

ABSTRACT: Our previous research has shown that α-(N)-heterocyclic thiosemicarbazone (TSC) metal complexes inhibit human topoisomerase IIα (TopoIIα), while the ligands without metals do not. To find out the structural elements of TSC that are important for inhibiting TopoIIα, we have synthesized two series of α-(N)heterocyclic TSCs with various substrate ring segments, side chain substitutions, and metal ions, and we have examined their activities in TopoIIα-mediated plasmid DNA relaxation and cleavage assays. Our goal is to explore the structure−activity relationship of α-(N)-heterocyclic TSCs and their effect on TopoIIα. Our data suggest that, similar to Cu(II)-TSCs, Pd(II)-TSC complexes inhibit plasmid DNA relaxation mediated by TopoIIα. In TopoIIα-mediated plasmid DNA cleavage assays, the Cu(II)-TSC complexes induce higher levels of DNA cleavage than their Pd(II) counterparts. The Cu(II)-TSC complexes with methyl, ethyl, and tert-butyl substitutions are slightly more effective than those with benzyl and phenyl groups. The α-(N)-heterocyclic ring substrates of the TSCs, including benzoylpyridine, acetylpyridine, and acetylthiazole, do not exhibit a significant difference in TopoIIα-mediated DNA cleavage. Our data suggest that the metal ion of TSC complexes plays a predominant role in inhibition of TopoIIα, the side chain substitution of the terminal nitrogen plays a secondary role, while the substrate ring segment has the least effect. Our molecular modeling data support the biochemical data, which together provide a mechanism by which Cu(II)-TSC complexes stabilize TopoIIαmediated cleavage complexes.

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INTRODUCTION Thiosemicarbazones (TSCs) are a group of compounds with an N−N−S tridentate coordination scaffold, which are excellent metal chelating agents. They were found to have antileukemic activities and antiproliferative activities against many cancer cell lines and transplanted murine tumors, making them promising candidates for chemotherapeutics.1,2 A special group, the α-(N)-heterocyclic TSCs, exhibits significant anticancer activities.3−5 One of the compounds from this group, 3-aminopyridine-2-carboxaldehyde TSC (Triapine) has been tested extensively in phase I/II clinical trials.6−9 The molecular mechanisms of how TSCs suppress tumor cell growth are quite complicated. Recent studies showed that an α-(N)-heterocyclic TSC compound, Dp44 mT, demonstrates selective activities against cancer cells attributed to inhibition of topoisomerase IIα (TopoIIα).10 Several other α(N)-heterocyclic TSCs and Cu(II)-TSC complexes also exhibit similar inhibition against TopoIIα.11−15 For example, quinoline-2-carboxaldehyde TSCs and their Cu(II) and Ni(II) complexes have been confirmed as TopoIIα inhibitors.11,13 © 2018 American Chemical Society

Other metal-TSC complexes including Pd(II), Pt(II), Ga(III), and Ru(II) were also explored in cancer cell studies and display anticancer activities, although the mechanism is unknown.1,16 Among those metals, Pd(II) and Pt(II) are of particular interest since their TSC complexes show comparable or higher anticancer activity to cisplatin, which is a widely prescribed antitumor drug.16−19 However, the molecular targets of Pd(II) and Pt(II) compounds have not been clarified. In addition, it is important to note that TSCs have been shown to impact the activity of multiple enzymes and possible to chelate metals, which adds to the complexity of examining the mechanism in cells.9,12,20 TopoIIα is an established target for anticancer agents. It is one of six topoisomerases found in humans and belongs to the type IIA topoisomerase subfamily. Type II topoisomerases generate a transient enzyme-bound double-stranded DNA break and pass an intact DNA segment through the break Received: July 26, 2018 Published: November 28, 2018 90

DOI: 10.1021/acs.chemrestox.8b00204 Chem. Res. Toxicol. 2019, 32, 90−99

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Chemical Research in Toxicology

other TSC compounds are listed in the Supporting Information. TSC compounds were dissolved in 100% DMSO and stored as 20 mM stock solutions at 4 °C. TopoIIα-Mediated Plasmid Cleavage Assay. Plasmid DNA cleavage assays were performed similarly to methods described previously.27 The reaction mixture contained 1 μg of human TopoIIα, 0.3 μg of pBR322 without or with 50 μM of compounds in a 20 μL reaction system with 10 mM Tris-HCl (pH 7.9), 5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, and 2.5% (v/v) glycerol. The reactions were initiated by addition of TopoIIα, incubated at 37 °C for 15 min, and stopped by addition of 2 μL of 5% of SDS and 2 μL of 250 mM EDTA. Proteinase K was added to the reaction and incubated for 30 min at 45 °C. The samples were then mixed with 2 μL of 6× agarose gel loading dye from Research Product International Corp. (Mount Prospect, IL) and subjected to electrophoresis in 1% TAE agarose gel with 0.5 μg/mL ethidium bromide at 110 V for 150 min. The results were then imaged with Bio-Rad Gel Doc XR+ or ChemiDoc MP imaging system. The data were quantified by Image Lab software from Bio-Rad (Hercules, California). TopoIIα-Mediated Plasmid Relaxation Assay. Plasmid DNA relaxation assays were carried out as described previously.27 Briefly, reactions were prepared with 0.2 μg of human TopoIIα, 0.3 μg of pBR322 and 1 mM ATP in 10 mM Tris (pH 7.9), 175 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, and 2.5% glycerol. TSC compounds with a concentration of 0.5, 1, 5, 10, or 50 μM were added. The reactions were incubated at 37 °C for 30 min and terminated by addition of 3 μL of stop solution with 77.5 mM EDTA and 0.77% SDS. To digest the proteins, the reactions were then incubated with 0.08 mg/mL proteinase K at 45 °C for 30 min. The samples were mixed with 2 μL of gel loading dye and run on a 1% agarose gel with TBE buffer. The gel was stained with 0.5 μg/mL ethidium bromide solution and visualized in Bio-Rad Gel Doc XR+ imaging system. Thin-Layer Chromatography-Based ATPase Assay. ATP hydrolysis was monitored using thin-layer chromatography (TLC) on a polyethylenimine (PEI) matrix (Merck KGaA, Darmstadt, Germany). Reactions were performed using conditions similar to relaxation reactions with minor adjustments. Reactions utilized DNA cleavage buffer with 1 mM ATP and were incubated at 37 °C, and 4 μL samples were drawn at 30 min and spotted on the TLC plate. Reactions were run in the absence (2% DMSO as a control) or presence of Cu(II)- or Pd(II)-TSC complexes, as noted in Figure 4. The plate was then placed in 400 mM ammonium carbonate inside the TLC chamber and resolved. Separation of ADP from ATP was imaged using an AlphaImager system (Santa Clara, CA) and quantified using AlphaImager software. Data are displayed relative to the quantified amount of ADP formed by TopoIIα at time = 30 min in the absence of drug. For plotting and comparison purposes, this value was normalized to 1. Molecular Docking. The structure of the ATPase domain of human type IIA DNA topoisomerase used for docking was from the Protein Data Bank (PDB). It was a dimer form, and the PDB ID was 1zxm.31 The structure was prepared by adding hydrogen atoms with the condition pH = 7.0, T = 300 K and a salt concentration of 0.1 M using the Protonate3D function of MOE 2018 (Chemical Computing Group Inc., Montreal, QC, Canada). The protein and the Cu(II)TSC compounds (without Cl− anion) were parametrized using the AMBER10:EHT force field in MOE. In particular, the protein parameters were taken from Amber ff99SB,32 while the ligand bonded parameters were obtained with 2D extended Hückel theory,33 the van der Waals (vdW) parameters were derived from general AMBER force field (GAFF),34 and the charges from bond charge increments. The receptor range in the protein was defined as all atoms of the following residues: R32, I33, Q35, R98, D152, D153, D154, and E155, as suggested by the previous study.13 Initial placement of the small ligand was done using the triangle matcher with London dG scoring and refined by Rigid Receptor docking using GBVI/WSA scoring. Five docked poses of each Cu(II)-TSC were obtained, and the one with lower score was used in the analysis.

before ligating the cleaved DNA. TopoIIα is a dimeric enzyme that requires Mg2+ to cleave and ligate DNA and ATP hydrolysis to perform DNA strand passage.20,21 In contrast to the persistent expression of the other human isoform topoisomerase IIβ, TopoIIα is expressed predominantly in proliferating cells and involved in resolving topological problems during DNA replication and cell division.22 Compounds targeting TopoIIα are divided into two broad categories: catalytic inhibitors and interfacial poisons.21,23−25 The catalytic inhibitors of TopoIIα generally inhibit ATP hydrolysis in the N-terminus of the enzyme. Interfacial poisons target the TopoIIα catalytic domain and convert transient double-stranded breaks to permanent breaks by blocking the ligation of cleaved DNA.23 Several research groups have examined the mechanism of how TSCs inhibit TopoIIα. Molecular modeling suggested that N,N-dimethyl-2-(quinolin-2-ylethylene) hydrazinecarbothioamide may be a catalytic inhibitor of TopoIIα by binding to the ATP site with higher affinity than ATP itself.12,26 Studies on quinoline-2-carboxaldehyde TSCs and their Cu(II) and Ni(II) complexes do not support a direct competition model because of the positive charge with the release of chloride after Cu(II)-TSC enters the cytoplasm.13 Our recent study showed that Cu(II) α-(N)-heterocyclic TSCs, Cu(II)-acetylpyridineethyl-TSC (CuYE), and Cu(II)-acetylpyrazine-methyl-TSC (CuZM) inhibit TopoIIα as noncompetitive catalytic inhibitors of ATP hydrolysis. We demonstrated both compounds inhibit ATP hydrolysis and increase DNA cleavage by TopoIIα.27 Our finding that Cu(II)-TSC acts as a noncompetitive TopoIIα catalytic inhibitor agrees with quinoline2-carboxaldehyde TSCs studies, which suggest that Cu(II)TSCs do not bind directly to the ATP binding site.13,27 Cu(II)-TSC complexes demonstrate higher inhibitory activity than their corresponding TSC ligands.11,13,15 To find out what structural elements are important for TSC inhibition of TopoIIα, we synthesized two series of TSCs without metal ions (referred to as ligands) and their Cu(II) and Pd(II) complexes. We also varied α-(N)-heterocyclic ring substrate segments, and side chain substitutions within the same series of α-(N)-heterocyclic TSCs. Those compounds were examined in TopoIIα-mediated plasmid DNA relaxation and cleavage assays, respectively. Additionally, we performed ATPase, ligation, molecular modeling, and ATP competition experiments. The following results explore the structure−activity relationship and clarify the mechanism of how TSC complexes stimulate TopoIIα-mediated DNA cleavage complexes.

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EXPERIMENTAL PROCEDURES

Enzyme and Materials. Human TopoIIα was expressed in yeast Saccharomyces cerevisiae and purified as described.27,28 TopoIIα was stored in 50 mM Tris pH 7.8, 750 mM KCl, 40% glycerol, and 0.5 mM dithiothreitol (DTT) as 1 mg/mL stock in liquid nitrogen. The recombinant plasmid pBR322 was amplified in Escherichia coli DH5α strain. The plasmid was purified following the protocol from Qiagen Plasmid Mega Kit and stored in ddH2O in liquid nitrogen as 1 mg/mL stock. Etoposide was purchased from Sigma and stored as 20 mM stock solutions in 100% dimethyl sulfoxide (DMSO) at 4 °C. Synthesis of Thiosemicarbazone Compounds. The benzoylpyridine-TSC (BZP)ligands and the Cu(II)-benzoylpyridine-TSC (CuBZP) complexes were synthesized by the method described previously.29 The synthesis of CuYE was described in previous studies.27 The synthesis of the acetylthiazole-TSC (ATZ) ligands was previously published.30 The synthesis and characterization of the 91

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Figure 1. Structures of α-(N)-heterocyclic TSCs with or without metal chelation. BZP and ATZ ligand only and Cu(II) and Pd(II) complex structures are shown. Additionally, Cu(II)-acetylpyridine-ethylthiosemicarbazone (CuYE) is shown on the right.

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RESULTS The two series of α-(N)-heterocyclic TSCs ligands in this study have different α-(N)-heterocyclic ring substrate segments: benzoylpyridine (BZP) and acetylthiazole (ATZ) as shown in Figure 1 and Table 1. Each ligand or metal complex also varied the side chain substitutions on the terminal nitrogen of the TSCs. Methyl, ethyl, tert-butyl, benzyl, and phenyl TSCs were used to form the series of ligands with each different α-(N)-heterocyclic ring substrate, to give 15 different ligands along with their Cu(II)-TSC and Pd(II)-TSC complexes (Supporting Information) (Table1). After synthesis and characterization, we examined their activities in TopoIIαmediated plasmid DNA relaxation assay and cleavage assay, respectively, to explore the structure−activity relationship of the TSC complexes and their inhibition on TopoIIα. And we further investigated the mechanism of why Cu(II)-TSCs increase TopoIIα-mediated DNA cleavage by molecular docking and ATP competition assays. The Pd(II)-TSC Complexes Inhibit TopoIIα-Mediated Plasmid Relaxation. The molecular targets of Pd(II)-TSC are unknown, although several Pd(II)-TSC complexes have been reported to exhibit anticancer activities.16,19,35 To examine whether Pd(II)-TSC complexes can inhibit TopoIIα

in a purified enzyme system, the compounds were tested in TopoIIα-mediated plasmid DNA relaxation assay. As shown in lanes 13−17 of Figure 2A, Pd(II)-BZP-TSCs (PdBZP) with various side chain substitutions inhibited TopoIIα-mediated relaxation at 10 μM. In Figure 2A, lanes 8−12, Cu(II)-BZPTSCs (CuBZP) inhibited TopoIIα-mediated DNA plasmid relaxation at 10 μM concentration, while ligand BZP series showed no inhibition. We are the first to show that Pd(II)TSC complexes inhibit TopoIIα. Also, our data are consistent with previous studies that Cu(II)-TSC complexes showed higher inhibition on TopoIIα than ligand TSCs.11,13,15,28 To further test if other Pd(II)-TSC complexes inhibit TopoIIα, we synthesized novel TSCs with five-membered thiazole ring structures such as ATZ (Supporting Information). As shown in Figure 2B, ATZ series compounds behave similarly to BZP complexes. Most of the Pd(II)-ATZ-TSCs (PdATZ) derivatives inhibited TopoIIα as presented in lanes 13−17 of Figures 2B with Pd(II)-ATZ-phenyl-TSCs (PdAP) exhibiting the weakest inhibition (lane 17 in Figure 2B). Ligand ATZs did not inhibit the activity of TopoIIα at 10 μM (lanes 3−7 in Figures 2B), but Cu(II)-ATZ-TSCs (CuATZ) with each side chain substitution showed strong inhibition in lanes 8−12 of Figure 2B. In summary, at the concentration of 92

DOI: 10.1021/acs.chemrestox.8b00204 Chem. Res. Toxicol. 2019, 32, 90−99

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Table 1. Acronyms of α-(N)-Heterocyclic Thiosemicarbazones and Their Activities in TopoIIα-Mediated DNA Cleavage benzoylpryridine thiosemicarbazone (BZP) benzoylpyridine methylthiosemicarbazone benzoylpyridine ethylthiosemicarbazone benzoylpyridine tertbutylthiosemicarbazone benzoylpyridine benzylthiosemicarbazone benzoylpyridine phenylthiosemicarbazone Cu(II) benzoylpyridine methylthiosemicarbazone Cu(II) benzoylpyridine ethylthiosemicarbazone Cu(II) benzoylpyridine tertbutylthiosemicarbazone Cu(II) benzoylpyridine benzylthiosemicarbazone Cu(II) benzoylpyridine phenylthiosemicarbazone Pd(II) benzoylpyridine methylthiosemicarbazone Pd(II) benzoylpyridine ethylthiosemicarbazone Pd(II) benzoylpyridine tertbutylthiosemicarbazone Pd(II) benzoylpyridine benzylthiosemicarbazone Pd(II) benzoylpyridine phenylthiosemicarbazone

acronym of BZP compounds with activity

acetylthiazole thiosemicarbazone (ATZ)

acronym of ATZ compounds with activity

BM (−) BE (−) BT (−)

acetylthiazole methylthiosemicarbazone acetylthiazole ethylthiosemicarbazone acetylthiazole tert-butylthiosemicarbazone

AM (−) AE (−) AT (−)

BB (−) BP (−) CuBM (+++)

acetylthiazole benzylthiosemicarbazone acetylthiazole phenylthiosemicarbazone Cu(II) acetylthiazole methylthiosemicarbazone Cu(II) acetylthiazole ethylthiosemicarbazone Cu(II) acetylthiazole tertbutylthiosemicarbazone Cu(II) acetylthiazole benzylthiosemicarbazone Cu(II) acetylthiazole phenylthiosemicarbazone Pd(II) acetylthiazole methylthiosemicarbazone Pd(II) acetylthiazole ethylthiosemicarbazone Pd(II) acetylthiazole tertbutylthiosemicarbazone Pd(II) acetylthiazole benzylthiosemicarbazone Pd(II) acetylthiazole phenylthiosemicarbazone

AB (−) AP (−) CuAM (+++)

CuBE (+++) CuBT (+++) CuBB (++) CuBP (++) PdBM (+) PdBE (+) PdBT (−) PdBB (−) PdBP (−)

CuAE (+++) CuAT (++) CuAB (++) CuAP (++) PdAM (+) PdAE (+) PdAT (−) PdAB (−) PdAP (−)

a Relative activity in parentheses: Relative DNA cleavage increase >7, +++; relative DNA cleavage increase 4−7, ++; relative DNA cleavage increase 2−4, +; relative DNA cleavage increase the substrate ring segments. Molecular modeling data (Figure 7) are consistent with results presented here since the substrate ring segments are pointing outside, while the terminal nitrogen substitutions are pointing toward the protein. Also, the ATP competition assay supports a model where Cu(II)-TSCs binds near but outside the ATP binding pocket. At this point, it is unclear why Cu(II)-TSCs result in higher levels of DNA cleavage than Pd(II)-TSCs. However, the data are consistent with previous studies that the Ni(II)-TSC complexes do not increase the DNA cleavage, while Cu(II)-TSCs do.13 One possibility is that the redox property of Cu may contribute to the high activity of Cu(II)-TSC complexes. Additional studies with other metal

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00204. Supplemental Methods: Plasmid DNA ligation assay supplemental methods: synthesis of thiosemicarbazone compounds. Figure S1: Concentration-dependent stimulation of TopoIIα-mediated plasmid DNA cleavage assay by CuAE, PdAE, and etoposide. Figure S2: Cu(II)etheyl-TSCs do not inhibit TopoIIα-mediated DNA ligation (PDF) 97

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(6) Kovacevic, Z., Chikhani, S., Lovejoy, D. B., and Richardson, D. R. (2011) Novel thiosemicarbazone iron chelators induce upregulation and phosphorylation of the metastasis suppressor N-myc down-stream regulated gene 1: a new strategy for the treatment of pancreatic cancer. Mol. Pharmacol. 80, 598−609. (7) Kovacevic, Z., Kalinowski, D. S., Lovejoy, D. B., Yu, Y., Suryo Rahmanto, Y., Sharpe, P. C., Bernhardt, P. V., and Richardson, D. R. (2011) The medicinal chemistry of novel iron chelators for the treatment of cancer. Curr. Top. Med. Chem. 11, 483−499. (8) Corce, V., Gouin, S. G., Renaud, S., Gaboriau, F., and Deniaud, D. (2016) Recent advances in cancer treatment by iron chelators. Bioorg. Med. Chem. Lett. 26, 251−256. (9) Kalinowski, D. S., Quach, P., and Richardson, D. R. (2009) Thiosemicarbazones: the new wave in cancer treatment. Future Med. Chem. 1, 1143−1151. (10) Rao, V. A., Klein, S. R., Agama, K. K., Toyoda, E., Adachi, N., Pommier, Y., and Shacter, E. B. (2009) The iron chelator Dp44mT causes DNA damage and selective inhibition of topoisomerase IIalpha in breast cancer cells. Cancer Res. 69, 948−957. (11) Zeglis, B. M., Divilov, V., and Lewis, J. S. (2011) Role of metalation in the topoisomerase IIalpha inhibition and antiproliferation activity of a series of alpha-heterocyclic-N4-substituted thiosemicarbazones and their Cu(II) complexes. J. Med. Chem. 54, 2391−2398. (12) Huang, H., Chen, Q., Ku, X., Meng, L., Lin, L., Wang, X., Zhu, C., Wang, Y., Chen, Z., Li, M., Jiang, H., Chen, K., Ding, J., and Liu, H. (2010) A series of alpha-heterocyclic carboxaldehyde thiosemicarbazones inhibit topoisomerase IIalpha catalytic activity. J. Med. Chem. 53, 3048−3064. (13) Bisceglie, F., Musiari, A., Pinelli, S., Alinovi, R., Menozzi, I., Polverini, E., Tarasconi, P., Tavone, M., and Pelosi, G. (2015) Quinoline-2-carboxaldehyde thiosemicarbazones and their Cu(II) and Ni(II) complexes as topoisomerase IIa inhibitors. J. Inorg. Biochem. 152, 10−19. (14) Bacher, F., Enyedy, E., Nagy, N. V., Rockenbauer, A., Bognar, G. M., Trondl, R., Novak, M. S., Klapproth, E., Kiss, T., and Arion, V. B. (2013) Copper(II) complexes with highly water-soluble L- and Dproline-thiosemicarbazone conjugates as potential inhibitors of Topoisomerase IIalpha. Inorg. Chem. 52, 8895−8908. (15) Conner, J., Medawala, W., Stephens, M., Morris, W., Deweese, J., Kent, P., Rice, J., Jiang, X., and Lisic, E. (2016) Cu(II) Benzoylpyridine Thiosemicarbazone Complexes: Inhibition of Human Topoisomerase IIα and Activity against Breast Cancer Cells. Open J. Inorg. Chem. 6, 146−154. (16) Kovala-Demertzi, D., Demertzis, M. A., Miller, J. R., Papadopoulou, C., Dodorou, C., and Filousis, G. (2001) Platinum(II) complexes with 2-acetyl pyridine thiosemicarbazone. Synthesis, crystal structure, spectral properties, antimicrobial and antitumour activity. J. Inorg. Biochem. 86, 555−563. (17) Matesanz, A. I., and Souza, P. (2007) Novel cyclopalladated and coordination palladium and platinum complexes derived from alpha-diphenyl ethanedione bis(thiosemicarbazones): structural studies and cytotoxic activity against human A2780 and A2780cisR carcinoma cell lines. J. Inorg. Biochem. 101, 1354−1361. (18) Kovala-Demertzi, D., Demertzis, M. A., Varagi, V., Papageorgiou, A., Mourelatos, D., Mioglou, E., Iakovidou, Z., and Kotsis, A. (1998) Antineoplastic and cytogenetic effects of platinum(II) and palladium(II) complexes with pyridine-2-carboxyaldehydethiosemicarbazone. Chemotherapy 44, 421−426. (19) Matesanz, A. I., Leitao, I., and Souza, P. (2013) Palladium(II) and platinum(II) bis(thiosemicarbazone) complexes of the 2,6diacetylpyridine series with high cytotoxic activity in cisplatin resistant A2780cisR tumor cells and reduced toxicity. J. Inorg. Biochem. 125, 26−31. (20) Wang, J. C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430−440. (21) Fortune, J. M., and Osheroff, N. (2000) Topoisomerase II as a target for anticancer drugs: when enzymes stop being nice. Prog. Nucleic Acid Res. Mol. Biol. 64, 221−253.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 931-372-3814. Fax: 931372-3434. *E-mail: [email protected], Phone: 615-966-7101. Fax: 615-966-7163. ORCID

Joseph E. Deweese: 0000-0001-9683-6723 Xiaohua Jiang: 0000-0002-0923-4482 Funding

This work was supported by funds from Faculty Development Grant from Tennessee Board of Regents (X.J. and E.C.L.), Faculty Research Grant of Tennessee Technological University (X.J.), Tennessee Technological University URECA! Grants Program (L.N. and A.R.B.), and the Lipscomb University College of Pharmacy and Health Sciences. J.T.W. was a participant in the Pharmaceutical Sciences Summer Research Program supported by the Lipscomb University College of Pharmacy and Health Sciences. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Xuanzhi Zhan and Jeffery Rice for helpful discussions. ABBREVIATIONS TSC, thiosemicarbazone; APY, acetylpyridine-thiosemicarbazone; BZP, benzoylpyridine-thiosemicarbazone; ATZ, acetylthiazole-thiosemicarbazone; MTSC or M, methyl-thiosemicarbazone; ETSC or E, ethyl-thiosemicarbazone; tBTSC or tB, tertbutyl-thiosemicarbazone; BzTSC or Bz, benzyl-thiosemicarbazone; PTSC or P, phenyl-thiosemicarbazone; PdYE, Pd(II)acetylpyridine-ethyl-thiosemicarbazone; CuBZP, Cu(II)-benzoylpyridine-thiosemicarbazone; PdBZP, Pd(II)-benzoylpyridine-thiosemicarbazone; CuATZ, Cu(II)-acetylthiazole-thiosemicarbazone; dATZ, Pd(II)-acetylthiazole-thiosemicarbazone; [Cu(APY-ETSC)Cl]: CuYE, Cu(II)-acetylpyridine-ethyl-thiosemicarbazone; [Cu(APZ-MTSC)Cl]: CuZM, Cu(II)-acetylpyrazine-methyl-thiosemicarbazone; ATP, adenosine-5′-triphosphate; DTT, dithiothreitol; DMSO, dimethyl sulfoxide

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REFERENCES

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DOI: 10.1021/acs.chemrestox.8b00204 Chem. Res. Toxicol. 2019, 32, 90−99

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DOI: 10.1021/acs.chemrestox.8b00204 Chem. Res. Toxicol. 2019, 32, 90−99