Cytotoxic effect and molecular docking of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide—a novel topoisomerase II inhibitor
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- Siwek, A., Stączek, P., Wujec, M. et al. J Mol Model (2013) 19: 1319. doi:10.1007/s00894-012-1679-6
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The preliminary cytotoxic effect of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride (1)—a potent topoisomerase II inhibitor—was measured using a MTT assay. It was found that the compound decreased the number of viable cells in both estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231breast cancer cells, with IC50 values of 146 ± 2 and 132 ± 2 μM, respectively. To clarify the molecular basis of the inhibitory action of 1, molecular docking studies were carried out. The results suggest that 1 targets the ATP binding pocket.
KeywordsThiosemicarbazide derivative Human topoisomerase II Cytotoxicity Molecular docking DFT calculation
An important chemotherapeutic target in the treatment of cancer is the ATP-dependent enzyme topoisomerase II (Topo II), which regulates the conformational changes in DNA topology necessary for transcription, replication, and chromosome condensation and segregation [1, 2]. Currently, six Topo II inhibitors (etoposide, teniposide, doxorubicin, daunorubicin, idarubicin, and mitoxantrone) are prescribed as highly anti-neoplastic drugs in clinical use. However, emerging tumor resistance and several side effects, such as hematological toxicity, nausea and vomiting, and hair loss, are associated with them [3, 4]. Therefore, efforts have been made by many research groups to find new chemicals with improved bioactivity. Although many compounds were found as cytostatic agents and Topo II and(or) I inhibitors, most showed significant toxicity for normal dividing cells, which precludes their use as drugs or lead molecule candidates . The discovery of new types of Topo II inhibitors that can be synthesized easily, show increased sensitivity in drug resistant tumors and decreased dose-limiting toxicities would be a significant addition to the choices available in the treatment of cancer.
Recently, as part of a search for the molecular basis of antibacterial activity of thiosemicarbazide-based compounds, we documented for the first time that one of the mechanisms of the bioactivity of these molecules is connected with inhibition of bacterial Topo IV [6, 7, 8]. Since both bacterial and human topoisomerases share a unique structural ATP-binding motif, called the Bergerat fold , we decided to conduct enzymatic studies to determine the effect of thiosemicarbazide derivatives on human topoisomerases. We found that the title compound, 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride (1) selectively inhibited Topo II activity almost completely at 15 μM concentration, proving more potent than etoposide under the same experimental conditions . To expand our initial findings with further details on the biological action pathways of 1, the compound was further assessed by preliminary cytotoxicity measurements. Herein we present the results of these investigations, as well as those of subsequent docking studies and DFT calculations, which allowed us to suggest that compound 1 targets the ATP binding pocket.
Materials and methods
Etoposide and camptothecin were purchased from TopoGEN (http://www.topogen.com/). Stock cultures of breast cancer MCF-7 and MDA-MB-231 were purchased from the American Type Culture Collection (Rockville, MD). Dulbecco’s minimal essential medium (DMEM) and foetal bovine serum (FBS) used in cell culture were products of Gibco (http://www.invitrogen.com/site/us/en/home/brands/Gibco.html). Glutamine, penicillin and streptomycin were obtained from Quality Biologicals (Gaithersburg, MD). [3H]-Thymidine (6.7 Ci/mmol) was the product of NEN (Boston, MA).
Human breast cancer MDA-MB-231 and MCF-7 cells maintained in DMEM supplemented with 10 % fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin at 37 °C. Cells were cultured in Costar flasks and subconfluent cells were detached with 0.05 % trypsin and 0.02 % EDTA in calcium-free phosphate buffered saline, counted in hemocytometers and plated at 5 × 105 cells per well in six-well plates (Nunc, http://www.nuncbrand.com) in 2 ml of growth medium (DMEM without phenol red with 10 % CPSR1). Cells reached about 80 % of confluency at day 3 and in most cases such cells were used for the assays.
To examine the effect of the studied drugs on MCF-7 and MDA-MB-231cell proliferation, the cells were seeded in 24-well tissue culture dishes at 1 × 105 cells/well with 1 ml growth medium. After 48 h (1.8 ± 0.1 × 105 cells/well) plates were incubated with varying concentrations of compound 1, chlorambucil and 0.5 μCi [3H]-thymidine for 24 h at 37 °C. Cells were rinsed three times with PBS, solubilised with 1 ml 0.1 M sodium hydroxide containing 1 % SDS, scintillation fluid (9 ml) was added and radioactivity incorporation into DNA was measured in a scintillation counter.
Cell viability assay
The assay was performed according to the method of Carmichael  using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Confluent cells, cultured for 24 h with various concentrations of the studied compounds in six-well plates were washed three times with PBS and then incubated for 4 h in 1 ml MTT solution (5 mg/ml PBS) at 37 °C in 5 % CO2 in an incubator. The medium was removed and 1 ml 0.1 M HCl in absolute isopropanol was added to attached cells. Absorbance of converted dye in living cells was measured at a wavelength of 570 nm. Cell viability of breast cancer cells cultured in the presence of ligands was calculated as a per cent of control cells.
Automated docking setup
Docking was performed by means of the FlexX program  as implemented in LeadIT software package  using models of Topo II binding site complexed with AMP-PNP (PDB id 1ZXN ) and etoposide (PDB id 3QX3 ) as templates. The ligand within the active site and all water molecules were removed while magnesium ion was allowed to remain with the charge of +2. The active site was defined to include all atoms within 6.5 Å radius of the native ligand. The first 100 top ranked docking poses were saved for each docking run. Subsequently, compounds 1–3 were docked using same docking parameters. Both rigid and flexible docking was performed (the results for the latter are presented). For compounds 1 and 3, both piperidine protonated and deprotonated forms were considered.
Following the methodology used previously  energies of best poses of compounds 1–4 obtained from FlexX docking were calculated using the B3LYP functional [17, 18] expressed in the basis set of 6–31 G(d) [19, 20, 21] as implemented in Gaussian 09  to describe their molecular properties. Natural population analysis (NPA) phase of NBO was used . The highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbitals were illustrated using the GaussView 5.0 program .
In all experiments, the mean values for six independent experiments ± standard deviations (SD) were calculated, unless otherwise indicated. The results were submitted to statistical analysis using Students t-test, accepting P < 0.05 as significant.
Results and discussion
Cytotoxic effect of 1
In this contribution we show compound 1 to be highly selective and potent inhibitor of human Topo II. From docking simulations, we conclude that its inhibitory action is connected with the ATP binding pocket although competitive inhibition assays are needed to confirm this hypothesis. These results will aid the rational design of novel thiosemicarbazide-based compounds that target the inhibition of Topo II and will provide insights into the discovery of novel anticancer agents.
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Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.