Antitumor activity of long-chain n-substituted 3-methylimidazolium ionic liquids against neuroblastoma cancer cells

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Antitumor activity of long-chain n-substituted 3-methylimidazolium ionic liquids against neuroblastoma cancer cells

Ivan V. Semenyuta

PhD, senior researcher of the biomedical research department V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry,

NAS of Ukraine, Ukraine

Diana M. Hodyna

PhD, senior researcher of the biomedical research department V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry,

NAS of Ukraine, Ukraine

Anastasia Gryniukova

PhD student of the biomedical research department

V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry,

NAS of Ukraine, Ukraine

Over the past twenty years, ionic liquids (ILs) have attracted the attention of the chemical and biological communities due to their extraordinary physical and chemical properties. Currently, these compounds are widely used in various fields of science, technology, and medicine [1]. Studies have shown that ILs cause oxidative stress, DNA damage, and apoptosis in mammals, insects, and others [2]. It is important to note that the cytotoxic effect of ILs depends not only on their chemical structure but also on the type of cell-biotarget because cancer cells differ from normal ones in specific molecular biotargets and metabolic pathways [3]. Today, the cytotoxic effect of various ILs has been shown on a large panel of cancer cells [4]. However, the exact molecular mechanism of their cytotoxic action remains discussion. neuroblastoma cancer currently

The potential anticancer activity of compounds (Table 1) was studied by cytotoxicity EC50 using cell lines obtained from the renowned American Type Culture Collection (ATCC). The investigation was performed on human neuroblast cells SK-N- DZ and, as reference cells, human fibroblasts MRC-5 and embryonic kidney HEK-293. The cytotoxic activity of compounds 1 -3 was analyzed in the range of concentrations (0 - 1000 pm) and references Doxorubicin (0 - 100 pm) and Cisplatin (0 - 300 pm). The CellTiter-Glo assay was used for the cytotoxicity study of all compounds, and the results are demonstrated in Table 1.

Table 1

The cytotoxicity activity of compounds 1 -3 as JAK2 inhibitors

¹- Cisplatin;² - Doxorubicin;³ - Abrocitinib; N\A - not activity

1-dodecyl-3-methylimidazolium chloride 1 has the highest cytotoxic effect (EC50=2.50 pm) on SK-N-DZ neuroblastoma cells (Table 1). Ester-functionalized derivatives 2 and 3 have lower effects with EC50 values of 65.60 pm and 15.80 pm, respectively; their cytotoxic activity is arranged in a row: 1 < 3 < 2. Their impact on the growth of non-cancer cells HEK293 and MRC-5 was less expressive. The compound's selectivity may be related to the overexpression of the oncogenic receptors on the neuroblastoma cells surface. So, interleukin 6 (IL6) and its receptor (IL6R) regulate cells growth and differentiation - they are expressed in 36% (IL6) and 81 % (IL6R) of the studied NB cell lines [5]. The IL6/JAK/STAT signaling pathway is key in the pathogenesis of NB, accordingly JAK2 kinase inhibitors are important antitumor agents. Thus, the JAK2 inhibitor - AZD1480 suppresses NB growth cells with EC50 = 0.36 pm [6]. Therefore, further studies focused on studying of JAK2 inhibitory effect of compounds 1-3. The ADP-Glo assay was used to study the JAK2 inhibition. Compounds were diluted to 0-100 pm concentrations, mixed with enzyme JAK2 (27.80 nm), and incubated at 25 °C (5 min). Enzyme activity was registered by the PHERAstar FSX reader (BMG Labtech) and was presented in Table

1. Table 1 data confirmed the JAK2 inhibition with compounds 2 and 3 of IC50 values of 36.16 pm and 16.33 pm, respectively (Abrocitinib [7] was used as a reference inhibitor).

The molecular docking method was used to study the potential mechanism of tyrosine kinase JAK2 inhibition. The preparation of ligand and protein was performed by the program AutoDock v.1.5.6[8]. The software ChemAxon Marvin Sketch

v.5.3.735 [9] was used to design the ligand structure, and the ligand structure optimization was performed using the MOPAC2016 program. The molecular docking was done using AutoDock Vina software v.1.1.2 [10], and the docking results were demonstrated by the Discovery Studio Visualizer v.4.0 [11]. The interaction features between ligand 3 and JAK2 [PDB ID: 7F7W] are shown in Figure 1.

Fig. 1. Molecular docking of compound 3 into the active site of JAK2 enzyme

Figure 1 introduces the formed ligand-JAK2 complex stabilized by five hydrogen bonds, electrostatic, and hydrophobic interactions. So, the imidazole ring constructs a hydrogen bond (3.54Л) with amino acids LEU551 and one hydrogen bond (2.35Л) and electrostatic interaction (2.83Л) with THR636. Also, the ligand carboxyl group forms three hydrogen bonds with amino acids THR636 (2.35Л), SER633 (2.36Л), and GLY632 (2.69Л). The C12 alkyl chain of compound 3 forms one hydrophobic interaction with amino acid ILE559 (4.00Л). T herefore, molecular docking results indicate the ligand-JAK2 complex formation by 1-dodecyloxycarbonylmethyl-3- imethyliimidazoliuim chloride and tyrosine kinase JAK2 with a predicted AG= -7.7 kcal/mol. The resulting complex is stabilized by hydrogen bonds, electrostatic interaction, and hydrophobic interactions with amino acid residues LEU551, THR636, SER633, GLY632, and ILE559.

The in vitro and in silico results suggest that long-chain N-substituted 3- methylimidazole derivatives have significant potential as anticancer agents with selectivity effect against neuroblastoma cells.

References:

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[2] Gonpalves, A. R., Paredes, X., Cristino, A. F., Santos, F. J. V., Queiros, C. S. (2021). Ionic liquids--A review of their toxicity to living organisms. International Journal of Molecular Sciences, 22(11), 5612.

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[5] Ara, T., Song, L., Shimada, H., Keshelava, N., Russell, H. V., Metelitsa, L. S., DeClerck, Y. A. (2009). Interleukin-6 in the bone marrow microenvironment promotes the growth and survival of neuroblastoma cells. Cancer research, 69(1), 329-337.

[6] Yan, S., Li, Z., & Thiele, C. J. (2013). Inhibition of STAT3 with orally active JAK inhibitor, AZD1480, decreases tumor growth in Neuroblastoma and Pediatric Sarcomas In vitro and In vivo. Oncotarget, 4(3), 433.

[7] George Abraham, B., Raivola, J., Virtanen, A., & Silvennoinen, O. (2020). Janus Kinase. Encyclopedia of Molecular Pharmacology, 1 -10.

[8] Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S., Olson, A. J. (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J.Comput.Chem., 30(16), 2785-91.

[9] ChemAxon, Marvin Sketch 5.3.735, https://www.chemaxon.com/ (accessed on March 27, 2024).

[10] Trott O, Olson AJ. (2010). AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry, 31 (2), 455-61.

[11] Dassault Systemes, Discovery Studio Visualizer, https://discover.3ds.com/ (accessed on March 28, 2024).

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