Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor

Abstract

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC₅₀ (0.37 ± 0.04 μM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 μM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.

Keywords: DNA intercalation; adenocarcinoma; copper(II) complex; indenoisoquinoline; molecular modeling; topoisomerase I.

Figure 1

Biologically active indenoisoquinolines.

Figure 2

Synthesis of the ligand WN191 and the Cu(II) complex WN198.

Figure 3

Molecular structure of Cu complex WN198 showing the local geometry around the copper and ligand. Selected bond lengths (Ǻ) and angles (°): Cu-N1BA 1.998(8); Cu-N4A 2.014(6); Cu-N1AA 1.990(7); Cu-O1W 1.980(7); Cu-O1D 2.333(7); N1BA Cu N4A 85.1(3); N1AA Cu N4A 86.2(3); O1W Cu N1AA 94.5(3); O1W Cu N1BA 94.6(3); N1BA Cu O1D 91.2(4); N4A Cu O1D 101.9(3); N1AA Cu O1D 88.1(3); O1W Cu O1D 94.0(3).

Figure 4

Non-cancerous cell viability under WN198 treatment. (A) MCF-10A IC₅₀ values are expressed as the mean ± SD of three independent experiments. Cisplatin and WN197 were used as control. (B) Xenopus oocyte G2/M transition was scored by the determination of a white spot at the animal pole showing the progression from prophase I to metaphase II (meiosis maturation) 12 h after microinjection and balneation with corresponding drugs (0.5, 1, 5 µM) and external stimulation by progesterone as a natural inducer (4 µM). Experiments were performed on 10 to 20 oocytes from three different females. (C) The viability of Xenopus embryos was followed after incubation with corresponding drugs (0.5, 1, 5 µM). Stages were identified using the Nieuwkoop and Faber table [24]: segmentation, (3 h 30 min after fertilization), gastrulation (9 h after fertilization), neurulation (19 h 45 min after fertilization), tailbud (24 h after fertilization). Experiments were performed on two independent fertilizations and 10 to 40 embryos in each condition.

Figure 5

WN191 and WN198 inhibited human topoisomerase I activity in a dose-dependent manner. Topoisomerase I (Top1) activity is determined by in vitro assays after the addition of either (A) WN198 or (B) WN191 at different concentrations (0.2, 0.5, 1, and 2 µM, lanes 5–8). Relaxed DNA (_RDNA, lane 1) or supercoiled DNA (_SCDNA, lane 2) is used as migration control. The Top1 activity control allowing the relaxation of _SCDNA is in lane 3. DMSO is the solvent control (5%, lane 4). _SCDNA is used in all other reactions in the presence of Top1. (C) Etoposide (VP-16, 50 µM; topoisomerase II (Top2) poison, lane 1) is the negative control of Top1 activity inhibition, and camptothecin (CPT, 10 µM; Top1 poison, lane 2) the positive control of Top1 activity inhibition. After topoisomerase reactions, DNA is run in a 1% agarose gel, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light.

Figure 6

Interaction models of the studied ligands and Top1. (A) Crystal structure of the topotecan poison (grey sticks) fitting the DNA groove (orange sticks) of Top1 (brown helices) (PDB ID: 1K4T, 2.1 Å). (B) The binding mode of WN170 (salmon sticks) is similar to topotecan’s binding mode. The main interaction is taking place inside the DNA groove. (C) The interaction model of WN191 (yellow sticks) is slightly different from WN170 as the larger branched arm has to switch down. The main part of the infarction is occurring through the aromatic ring. (D) The binding mode of WN198 (pink sticks with the copper molecule as a blue ball) is similar to the binding mode of WN191.

Figure 7

WN198-induced autophagy. MDA-MB-231, HeLa, and HT-29 cell lines were treated for 24 h with doxorubicin (Dox, 5 µM), rapamycin (Rap, 0.5 µM), or WN198 (0.5, 1, 5 µM). (A) Western blots were performed with anticleaved caspase 3 and anticleaved PARP antibodies for detection of apoptosis. Anti-γH2AX antibody was used for detection of DNA breaks. (B) Western blot analysis of Beclin-1, and LC3 markers were used for detection of autophagy. LC3-II/LC3-I ratio was determined (arrow). β-actin level was used as a loading control. Relative protein levels were expressed by densitometry using software (Fiji Software, v1.52i).