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. 2023 Feb 25;16(3):353.
doi: 10.3390/ph16030353.

5,8-Dimethyl-9 H-carbazole Derivatives Blocking hTopo I Activity and Actin Dynamics

Jessica Ceramella  1 , Domenico Iacopetta  1 , Anna Caruso  1 , Annaluisa Mariconda  2 , Anthi Petrou  3 , Athina Geronikaki  3 , Camillo Rosano  4 , Carmela Saturnino  2 , Alessia Catalano  5 , Pasquale Longo  6 , Maria Stefania Sinicropi  1
Affiliations

5,8-Dimethyl-9 H-carbazole Derivatives Blocking hTopo I Activity and Actin Dynamics

Jessica Ceramella et al. Pharmaceuticals (Basel). .

Abstract

Over the years, carbazoles have been largely studied for their numerous biological properties, including antibacterial, antimalarial, antioxidant, antidiabetic, neuroprotective, anticancer, and many more. Some of them have gained great interest for their anticancer activity in breast cancer due to their capability in inhibiting essential DNA-dependent enzymes, namely topoisomerases I and II. With this in mind, we studied the anticancer activity of a series of carbazole derivatives against two breast cancer cell lines, namely the triple negative MDA-MB-231 and MCF-7 cells. Compounds 3 and 4 were found to be the most active towards the MDA-MB-231 cell line without interfering with the normal counterpart. Using docking simulations, we assessed the ability of these carbazole derivatives to bind human topoisomerases I and II and actin. In vitro specific assays confirmed that the lead compounds selectively inhibited the human topoisomerase I and interfered with the normal organization of the actin system, triggering apoptosis as a final effect. Thus, compounds 3 and 4 are strong candidates for further drug development in multi-targeted therapy for the treatment of triple negative breast cancer, for which safe therapeutic regimens are not yet available.

Keywords: actin dynamics; anticancer; docking simulation; human topoisomerases I/II.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of Ellipticine and the studied carbazole derivatives (1–5) previously synthesized by us [25].
Figure 2
Figure 2
The three-dimensional structure of the human proteins Topoisomerase I (Panel A), Topoisomerase II (Panel B) and Actin (Panel C) bound to compounds 3 and 4 are drawn. Proteins are schematically reported as ribbons. Ligands binding poses are described as colored sticks.
Figure 3
Figure 3
(a) hTopo I supercoiled relaxing activity. hTopo I was exposed to the vehicle alone (DMSO, lane 2) or compounds 3 and 4 at the concentration of 1 μM (lanes 3 and 4). Then, the hTopo I reaction products were visualized on agarose gel. Supercoiled DNA (plasmid pHOT1) was used as marker (lane 1). (b) hTopo II decatenation assay. hTopo II was exposed to the vehicle alone (DMSO, lane 3) or compounds 3 and 4 at the concentrations of 1 μM (lanes 4 and 5) and 10 μM (lanes 6 and 7). Then, the hTopo II reaction products were visualized on agarose gel. Decatenated DNA and kinetoplast DNA (kDNA) were used as markers (lanes 1 and 2).
Figure 4
Figure 4
Actin immunofluorescence studies. MDA-MB-231 cells were exposed for 24 h to the vehicle alone (CTRL), 0.1 μM LA or carbazole derivatives 3 and 4 (used at their IC50 values). Then the cells were further processed, as indicated in the experimental section. The inverted fluorescence microscope was adopted to observe and image all the immunofluorescence figures (40× magnification). Panels A: nuclear stain with DAPI (λex/λem = 350/460 nm); Panels B: β-actin (Alexa Fluor® 568; λex/λem = 644/665 nm); Panels C show a merge. Representative fields are reported.
Figure 5
Figure 5
(A) In vitro actin polymerization assay. Compounds 3 and 4 (used at the concentration of 5 µM) were incubated with the labeled rabbit muscle actin in order to verify their ability to inhibit the protein polymerization. (B) In vitro actin depolymerization assay. After actin polymerization, compounds 3 and 4 (5 µM) were added to the reaction mixture, in order to determine their ability to act as depolymerizing agents. For both the assays, the vehicle DMSO was used as a negative control. Actin-targeting agents, LA and CB, both at the concentration of 5 μM, were used as positive controls. The assemblage of the actin filaments was established by monitoring the fluorescence (λEx/Em= 365/410 nm) in kinetic mode for 1 h at room temperature by using a microplate reader. The graphics are representative of three separate experiments and error bars represent the standard deviations.
Figure 6
Figure 6
Tunel Assay. MDA-MB-231 breast cancer cells were treated with compounds 3 and 4 at the concentration equal to their IC50 values or with vehicle (CTRL) for 24 h. Then they were exposed to the TdT enzyme, further processed (see experimental section for more details) and visualized under a fluorescence microscope (20× magnification). Panels A: DAPI, λex/em= 350 nm/460 nm. Panels B: CFTM488 A, λex/em = 490 nm/515 nm. Panels C show the overlay channels.
Figure 7
Figure 7
(A) BOILED-Egg diagram for all the designed compounds. (B) Bioavailability radar chart of all compounds. The pink area represents the optimal range for each property for oral bioavailability, Lipophilicity (LIPO): XLOGP3 between −0.7 and +5.0, Molecular weight (SIZE): MW between 150 and 500 g/mol, Polarity (POLAR) TPSA between 20 and 130 Å2, Solubility (INSOLU): log S not higher than 6, Saturation (INSATU): fraction of carbons in the sp3 hybridization not less than 0.25, and Flexibility (FLEX): no more than 9 rotatable bonds.
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References

    1. Gluszynska A. Biological potential of carbazole derivatives. Eur. J. Med. Chem. 2015;94:405–426. doi: 10.1016/j.ejmech.2015.02.059. - DOI - PubMed
    1. Caruso A., Chimento A., El-Kashef H., Lancelot J.-C., Panno A., Pezzi V., Saturnino C., Sinicropi M.S., Sirianni R., Rault S. Antiproliferative activity of some 1,4-dimethylcarbazoles on cells that express estrogen receptors: Part I. J. Enzym. Inhib. Med. Chem. 2012;27:609–613. doi: 10.3109/14756366.2011.603132. - DOI - PubMed
    1. Caruso A., Voisin-Chiret A.S., Lancelot J.C., Sinicropi M.S., Garofalo A., Rault S. Efficient and simple synthesis of 6-aryl-1,4-dimethyl-9H-carbazoles. Molecules. 2008;13:1312–1320. doi: 10.3390/molecules13061312. - DOI - PMC - PubMed
    1. Asche C., Demeunynck M. Antitumor carbazoles. Anticancer Agents Med. Chem. 2007;7:247–267. doi: 10.2174/187152007780058678. - DOI - PubMed
    1. Bjornsti M.A., Kaufmann S.H. Topoisomerases and cancer chemotherapy: Recent advances and unanswered questions. F1000Research. 2019;8:F1000 Faculty Rev-1704. doi: 10.12688/f1000research.20201.1. - DOI - PMC - PubMed

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