Elsevier

Bioorganic Chemistry

Volume 82, February 2019, Pages 74-85
Bioorganic Chemistry

Pentamethinium salts as ligands for cancer: Sulfated polysaccharide co-receptors as possible therapeutic target

https://doi.org/10.1016/j.bioorg.2018.02.011Get rights and content

Highlights

  • Synthesis of pentamethinium salts with benzothiazolium and indolium side units.

  • Affinity to sulfated polysaccharides correlated with their biological activity.

  • Side heteroaromates responsible for cytotoxicity and selectivity to cancer cells.

  • Charged salts with benzothiazole high efficacy against taxol-resistant cancer cells.

Abstract

A series of pentamethinium salts with benzothiazolium and indolium side units comprising one or two positive charges were designed and synthesized to determine the relationships among the molecular structure, charge density, affinity to sulfated polysaccharides, and biological activity. Firstly, it was found that the affinity of the pentamethinium salts to sulfated polysaccharides correlated with their biological activity. Secondly, the side heteroaromates displayed a strong effect on the cytotoxicity and selectivity towards cancer cells. Finally, doubly charged pentamethinium salts possessing benzothiazolium side units exhibited remarkably high efficacy against a taxol-resistant cancer cell line.

Introduction

Cell-cell communication is essential for multicellular organisms. Every cellular process, including cell growth, death, and differentiation, depends on the activity and pathways of signaling molecules. Aberrant changes in the system of cell-to-cell communication may result in various pathological states, such as cancer, chronic inflammatory disorders, and autoimmune diseases [1]. The involved pathological signaling pathways seem to represent attractive targets for a therapeutic intervention.

A common way to modulate, inhibit or activate cell-to-cell communication is based on exploitation of specific chemical ligands targeting individual components of the signaling pathways, such as signaling molecules and their corresponding receptors. However, the therapeutic strategy affecting only one target has not proved to be very effective and often resulted in resistance. At present, a new concept involving molecules interfering simultaneously with multiple targets is being explored as a more efficient strategy [2], [3], [4]. Such an approach might enable low-dose administration of active agents resulting in elimination of possible undesired adverse effects and producing additional synergistic or additive therapeutic results. The design of multi-targeted chemical ligands should be based on a chemical structure of their respective targets. Therefore, the structure of growth factor receptors commonly overexpressed on the surface of cancer cells should be the main clue for the design of rational synthetic ligands. However, due to a very complex nature of these protein receptors, it is very difficult to achieve their identification by specific recognition and targeting. Importantly, several reports indicated that the saccharide parts of the growth factor receptors, sulfated glycosaminoglycans (GAG), are essential for the receptor function on cancer cells. This fact was favorably exploited for their detection and recognition [5], [6], [7] and directed our design of multitargeted ligands by focusing on polysulfated GAGs.

Polysulfated GAGs (e.g. heparan sulfate, chondroitin sulfate) are polysaccharides with high structural variability, possessing a negative charge due to sulfate and carboxyl groups. The average number of GAG modifications is one sulfate and one carboxyl group per one repeating disaccharide unit. The GAGs are functional, long unbranched heteropolysaccharides playing a key role in regulation of a number of cellular events as well as physiological and pathological processes [8]. Their biological function generally depends on the interactions of the binding domain (highly sulfated part of GAGs) with signal factors (cytokines and soluble growth factors) [9]. Changes in GAGs overexpression undoubtedly influence proliferation of cancer cells, metastasis, and tumor progression [10], [11] Therefore, targeted inhibition of the expression [12] or glycosylation [13] of these co-receptors can lead to reduction of the tumorigenic phenotype, metastatic potential and angiogenesis. Polysulfated GAGs also play a role in other serious pathophysiological settings, including amyloid diseases (Alzheimer’s disease; serum amyloid A protein, transthyretin-related amyloidosis and amyloid light-chain amyloidosis, type 2 diabetes, Parkinson’s disease, amyotrophic lateral sclerosis and prion diseases) [14], infectious diseases (severe anemia, respiratory distress, cerebral ischemia and AIDS) [15], [16], [17], [18], inflammatory conditions and some developmental disorders [19], [20]. In the amyloid genesis, sulfated GAGs bind amyloid proteins and contribute to their aggregation in a fibrillar insoluble form [21]. They also protect the formed aggregates against proteolysis and probably facilitate stimulation of nitric oxide production and tumor necrosis factor alpha (TNF-α) expression [22], [23]. Even though the amyloid proteins are structurally unrelated, they share the ability to bind sulfated GAGs, which could be envisaged as a potential therapeutic target in amyloid genesis. Several groups reported positive correlation of the sulfation degree of the used polysaccharides with the change of their conformation states [24], [25], [26] thereby indicating the importance of the sulfated part of GAGs. In agreement with this hypothesis, a higher level of sulfated GAGs was observed in human brains (post-mortem) affected by amyloid diseases [22], [27].

On the other hand, sulfated GAGs can serve as therapeutic agents for treatment of cancer, viral and bacterial infections, or blood clotting [28], [29], [30]. All these facts indicate a high clinical potential of sulfated GAGs, which could be specifically recognized and targeted through their highly sulfated domains. To our best knowledge, the detection of sulfated polysaccharide receptors is an unexplored area. In theory, a suitable tool for recognition of anionic polymers, such as GAGs, might be cationic ligands. Furthermore, the structural motif for GAG-specific ligands should also include, beside the cationic charge motif (binding the anionic sulfate group), heteroaromatic groups (binding the saccharide part) [31]. Interestingly, our team also achieved recognition of heparan sulfate by generating bile acid porphyrin conjugates [6]. The inspiration for this approach arose from already reported sensors recognizing an extensively sulfated polysaccharide, heparin [32], [33], [34].

Previously, we reported a significant spectral response of the pentamethinium structural motif in the presence of sulfated polysaccharides (namely heparin) [35]. However, the hydrophobicity of pentamethinium salts and their strong aggregation in aqueous solutions required admixture of organic solvents negatively influencing the desired interactions. Therefore, in this work we aimed to develop chemical ligands based on the pentamethinium system with high selectivity towards the sulfated polysaccharides in aqueous media at pH 7.34 allowing applicability at physiological conditions. Our goal was to confirm the hypothesis that the affinity of chemical ligands consisting of pentamethinium salts correlates with the amount of anionic sulfate groups in the studied analytes resembling cancer receptors. The issue of the pentamethinium system hydrophobicity [35], [36] required redesign of the structure to ensure sufficient hydrophilicity, and, additionally, charge variability of the whole system. The most frequently used method for enhancing hydrophilicity of largely hydrophobic compounds is substitution with alkyl sulfate groups [37], [38], [39], but this approach was not feasible due to possible interferences with the tested analytes. Our strategy to achieve higher hydrophilicity and to increase charge density of the pentamethinium system was based on the introduction of a second positive charge by nitrogen quarternization. Some of γ-substituted pentamethinium salts described in our previous work contained a pyridyl substituent in the meso-position [40], the presence of which offers the possibility of nitrogen quarternization, and therefore these salts were included in the presented study.

Section snippets

Results and discussion

The aim of this work was to study and compare monocationic and corresponding dicationic pentamethinium salts, and to elucidate the influence of the second positive charge and a side heteroaromatic unit (benzothiazolium or dimethylindolium) on their biological properties. Our strategy was based on the selection of structures that could be quarternized to the second stage by a methyl group. The most feasible way to do this is utilization of pentamethinium salts substituted in γ-position by a

Conclusions

In summary, we designed and synthesized a group of pentamethinium salts, 14, with one or two positive charges and assayed them for recognition of sulfated polysaccharides. Based on the obtained results, we conclude that the synthetic ligands, mainly salt 2 with side benzothiazolium units, can be effectively used for recognition of sulfated polysaccharides. We found out that the higher number of sulfate groups together with increased density of anionic charge of sulfated polysaccharides

Synthesis of compound 1

The flask was charged with 2-(4-pyridyl)malondialdehyde (150 mg, 1.0 mmol), 2-methyl-3-propyl benzothiazolium iodide (640 mg, 20.1 mmol) and dry n-butanol (25 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the mixture was filtered. The solid was washed with ethanol (3 · 5 mL) and dried in vacuum. Product 1 was obtained as green powder, 498 mg, 79%. 1H NMR (300 MHz, DMSO‑d6, 25 °C): 8.94 (2H, d, J = 6.4 Hz), 8.14–7.80 (8H, m), 7.60 (2H, t, J = 8.2 Hz),

Acknowledgements

This work was supported by National Sustainability Program II [Project BIOCEV-FAR; LQ1604]; Project “BIOCEV” [CZ.1.05/1.1.00/02.0109]; Charles University in Prague [UNCE 204064, Progress Q26/LF1 and Q27/LF1]; and Specific University Research [MSMT-SVV-260367, MSMT No 20-SVV/2017 and JIGA 2017 UCT grant 2017]. Further, this work was supported by the Ministry of Education, Youth and Sports of CR LO 1304, partially also by “EATRIS-CZ” (LM2015064) and by the Grant Agency of the Czech Republic

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