In vitro biological evaluation of a novel folic acid-targeted receptor quantum dot-ß-cyclodextrin carrier for C-2028 unsymmetrical bisacridine in the treatment of human lung and prostate cancers.

BACKGROUND: Traditional small-molecule chemotherapeutics usually do not distinguish tumors from healthy tissues. However, nanotechnology creates nanocarriers that selectively deliver drugs to their site of action. This work is the next step in the development of the quantum dot-ß-cyclodextrin-folic acid (QD-ß-CD-FA) platform for targeted and selected delivery of C-2028 unsymmetrical bisacridine in cancer therapy. METHODS: Herein, we report an initial biological evaluation (using flow cytometry and light microscopy) as well as cell migration analysis of QD-ß-CD(C-2028)-FA nanoconjugate and its components in the selected human lung and prostate cancer cells, as well as against their respective normal cells. RESULTS: C-2028 compound induced apoptosis, which was much stronger in cancer cells compared to normal cells. Conjugation of C-2028 with QDgreen increased cellular senescence, while the introduction of FA to the conjugate significantly decreased this process. C-2028 nanoencapsulation also reduced cell migration. Importantly, QDgreen and QDgreen-ß-CD-FA themselves did not induce any toxic responses in studied cells. CONCLUSIONS: In conclusion, the results demonstrate the high potential of a novel folic acid-targeted receptor quantum dot-ß-cyclodextrin carrier (QDgreen-ß-CD-FA) for drug delivery in cancer treatment. Nanoplatforms increased the amount of delivered compounds and demonstrated high suitability.

Targeting yeast topoisomerase II by imidazo and triazoloacridinone derivatives resulting in their antifungal activity.

Fungal pathogens are considered as serious factors for deadly diseases and are a case of medical concern. Invasive fungal infections also complicate the clinical course of COVID-19, leading to a significant increase in mortality. Furthermore, fungal strains' multidrug resistance has increased the demand for antifungals with a different mechanism of action. The present study aimed to identify antifungal compounds targeting yeast topoisomerase II (yTOPOII) derived from well-known human topoisomerase II (hTOPOII) poisons C-1305 and C-1311. Two sets of derivatives: triazoloacridinones (IKE1-8) and imidazoacridinones (IKE9-14) were synthetized and evaluated with a specific emphasis on the molecular mechanism of action. Our results indicated that their effectiveness as enzyme inhibitors was not solely due to intercalation ability but also as a result of influence on catalytic activity by the formation of covalent complexes between plasmid DNA and yTOPOII. Lysine conjunction increased the strength of the compound's interaction with DNA and improved penetration into the fungal cells. Triazoloacridinone derivatives in contrast to starting compound C-1305 exhibited moderate antifungal activity and at least twice lower cytotoxicity. Importantly, compounds (IKE5-8) were not substrates for multidrug ABC transporters whereas a derivative conjugated with lysine (IKE7), showed the ability to overcome C. glabrata fluconazole-resistance (MIC 32-64 µg mL-1).

Cellular Effects of Selected Unsymmetrical Bisacridines on the Multicellular Tumor Spheroids of HCT116 Colon and A549 Lung Cancer Cells in Comparison to Monolayer Cultures.

Multicellular tumor spheroids are a good tool for testing new anticancer drugs, including those that may target cancer stem cells (CSCs), which are responsible for cancer progression, metastasis, and recurrence. Therefore, we applied this model in our studies of highly active antitumor unsymmetrical bisacridines (UAs). We investigated the cellular response induced by UAs in 2D and 3D cultures of HCT116 colon and A549 lung cancer cells, with an additional focus on their impact on the CSC-like population. We showed that UAs affected the viability of the studied cells, as well as their spherogenic potential in the 2D and 3D cultures. Furthermore, we proved that the most promising UAs (C-2045 and C-2053) induced apoptosis in the HCT116 and A549 spheres to a similar, or even higher, extent than what was found in monolayer conditions. Next, we identified the population of the CSC-like cells in the 2D and 3D cultures of the studied cell lines by determining the levels of CD166, CD133, CD44, and EpCAM markers. We showed that the selected UAs affected the CSC-like population in both of the cell lines, and that A549 was affected more profoundly in 3D than in 2D cultures. Thus, the UAs exhibited high antitumor properties in both the 2D and 3D conditions, which makes them promising candidates for future therapeutic applications.

In Vitro Enzyme Kinetics and NMR-Based Product Elucidation for Glutathione S-Conjugation of the Anticancer Unsymmetrical Bisacridine C-2028 in Liver Microsomes and Cytosol: Major Role of Glutathione S-Transferase M1-1 Isoenzyme.

This work is the next step in studying the interplay between C-2028 (anticancer-active unsymmetrical bisacridine developed in our group) and the glutathione S-transferase/glutathione (GST/GSH) system. Here, we analyzed the concentration- and pH-dependent GSH conjugation of C-2028 in rat liver microsomes and cytosol. We also applied three recombinant human GST isoenzymes, which altered expression was found in various tumors. The formation of GSH S-conjugate of C-2028 in liver subfractions followed Michaelis-Menten kinetics. We found that C-2028 was conjugated with GSH preferentially by GSTM1-1, revealing a sigmoidal kinetic model. Using a colorimetric assay (MTT test), we initially assessed the cellular GST/GSH-dependent biotransformation of C-2028 in relation to cytotoxicity against Du-145 human prostate cancer cells in the presence or absence of the modulator of GSH biosynthesis. Pretreatment of cells with buthionine sulfoximine resulted in a cytotoxicity decrease, suggesting a possible GSH-mediated bioactivation process. Altogether, our results confirmed the importance of GSH conjugation in C-2028 metabolism, which humans must consider when planning a treatment strategy. Finally, nuclear magnetic resonance spectroscopy elucidated the structure of the GSH-derived product of C-2028. Hence, synthesizing the compound standard necessary for further advanced biological and bioanalytical investigations will be achievable.

Electrochemistry/mass spectrometry (EC/MS) for fast generation and identification of novel reactive metabolites of two unsymmetrical bisacridines with anticancer activity.

The development of a new drug requires knowledge about its metabolic fate in a living organism, regarding the comprehensive assessment of both drug therapeutic activity and toxicity profiles. Electrochemistry (EC) coupled with mass spectrometry (MS) is an efficient tool for predicting the phase I metabolism of redox-sensitive drugs. In particular, EC/MS represents a clear advantage for the generation of reactive drug transformation products and their direct identification compared to biological matrices. In this work, we focused on the characterization of novel electrochemical products of two representative unsymmetrical bisacridines (C-2028 and C-2045) with demonstrated high anticancer activity. The electrochemical thin-layer flow-through cell µ-PrepCell 2.0 (Antec Scientific) was used here for the effective metabolite electrosynthesis. The electrochemical simulation of C-2028 reductive and C-2045 oxidative metabolism resulted in the generation of new products that were not observed before. The formation of nitroso [M-O+H]+ and azoxy [2M-3O+H]+ species from C-2028, as well as a series of hydroxylated and/or dehydrogenated products, including possible quinones [M-2H+H]+ and [M+O-2H+H]+ from C-2045, was demonstrated. For the latter, a glutathione S-conjugate (m/z 935.3130) was also obtained in measurements supplemented with the excess of reduced glutathione. For the identification of the products of interest, structural confirmation based on MS/MS fragmentation experiments was performed. Novel products of electrochemical conversions of unsymmetrical bisacridines were discussed in the context of their possible biological effect on the human organism.

The interactions of monomeric acridines and unsymmetrical bisacridines (UAs) with DNA duplexes: an insight provided by NMR and MD studies.

Members of a novel class of anticancer compounds, exhibiting high antitumor activity, i.e. the unsymmetrical bisacridines (UAs), consist of two heteroaromatic ring systems. One of the ring systems is an imidazoacridinone moiety, with the skeleton identical to the structural base of Symadex. The second one is a 1-nitroacridine moiety, hence it may be regarded as Nitracrine's structural basis. These monoacridine units are connected by an aminoalkyl linker, which vary in structure. In theory, these unsymmetrical dimers should act as double-stranded DNA (dsDNA) bis-intercalators, since the monomeric units constituting the UAs were previously reported to exhibit an intercalating mode of binding into dsDNA. On the contrary, our earlier, preliminary studies have suggested that specific and/or structurally well-defined binding of UAs into DNA duplexes might not be the case. In this contribution, we have revisited and carefully examined the dsDNA-binding properties of monoacridines C-1305, C-1311 (Symadex), C-283 (Ledakrin/Nitracrine) and C-1748, as well as bisacridines C-2028, C-2041, C-2045 and C-2053 using advanced NMR techniques, aided by molecular modelling calculations and the analysis of UV-VIS spectra, decomposed by chemometric techniques. These studies allowed us to explain, why the properties of UAs are not a simple sum of the features exhibited by the acridine monomers.

pH-Responsive Drug Delivery Nanoplatforms as Smart Carriers of Unsymmetrical Bisacridines for Targeted Cancer Therapy.

Selective therapy and controlled drug release at an intracellular level remain key challenges for effective cancer treatment. Here, we employed folic acid (FA) as a self-navigating molecule in nanoconjugates containing quantum dots (QDs) and ß-cyclodextrin (ß-CD) for the delivery of antitumor unsymmetrical bisacridine compound (C-2028) to lung and prostate cancers as well as normal cells. The bisacridine derivative can form the inclusion complex with ß-cyclodextrin molecule, due to the presence of a planar fragment in its structure. The stability of such a complex is pH-dependent. The drug release profile at different pH values and the mechanism of C-2028 release from QDs-ß-CD-FA nanoconjugates were investigated. Next, the intracellular fate of compounds and their influence on lysosomal content in the cells were also studied. Confocal Laser Scanning Microscopy studies proved that all investigated compounds were delivered to acidic organelles, the pH of which promoted an increased release of C-2028 from its nanoconjugates. Since the pH in normal cells is higher than in cancer cells, the release of C-2028 from its nanoconjugates is decreased in these cells. Additionally, we obtained the concentration profiles of C-2028 in the selected cells treated with unbound C-2028 or nanoconjugate by the HPLC analysis.

Foliate-Targeting Quantum Dots-ß-Cyclodextrin Nanocarrier for Efficient Delivery of Unsymmetrical Bisacridines to Lung and Prostate Cancer Cells.

Targeted drug delivery by nanocarriers molecules can increase the efficiency of cancer treatment. One of the targeting ligands is folic acid (FA), which has a high affinity for the folic acid receptors, which are overexpressed in many cancers. Herein, we describe the preparation of the nanoconjugates containing quantum dots (QDs) and ß-cyclodextrin (ß-CD) with foliate-targeting properties for the delivery of anticancer compound C-2028. C-2028 was bound to the nanoconjugate via an inclusion complex with ß-CD. The effect of using FA in QDs-ß-CD(C-2028)-FA nanoconjugates on cytotoxicity, cellular uptake, and the mechanism of internalization in cancer (H460, Du-145, and LNCaP) and normal (MRC-5 and PNT1A) cells was investigated. The QDs-ß-CD(C-2028)-FA were characterized using DLS (dynamic light scattering), ZP (zeta potential), quartz crystal microbalance with dissipation (QCM-D), and UV-vis spectroscopy. The conjugation of C-2028 with non-toxic QDs or QDs-ß-CD-FA did not change the cytotoxicity of this compound. Confocal microscopy studies proved that the use of FA in nanoconjugates significantly increased the amount of delivered compound, especially to cancer cells. QDgreen-ß-CD(C-2028)-FA enters the cells through multiple endocytosis pathways in different levels, depending on the cell line. To conclude, the use of FA is a good self-navigating molecule in the QDs platform for drug delivery to cancer cells.

The Effect of Conjugation with Octaarginine, a Cell-Penetrating Peptide on Antifungal Activity of Imidazoacridinone Derivative.

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 µg mL-1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect.

Photoresponsive Amide-Based Derivatives of Azobenzene-4,4'-Dicarboxylic Acid-Experimental and Theoretical Studies.

Azobenzene derivatives are one of the most important molecular switches for biological and material science applications. Although these systems represent a well-known group of compounds, there remains a need to identify the factors influencing their photochemical properties in order to design azobenzene-based technologies in a rational way. In this contribution, we describe the synthesis and characterization of two novel amides (L1 and L2) containing photoresponsive azobenzene units. The photochemical properties of the obtained compounds were investigated in DMSO by UV-Vis spectrophotometry, as well as 1H NMR spectroscopy, and the obtained results were rationalized via Density Functional Theory (DFT) methods. After irradiation with UV light, both amides underwent trans to cis isomerization, yielding 40% and 22% of the cis isomer of L1 and L2 amides, respectively. Quantum yields of this process were determined as 6.19% and 2.79% for L1 and L2, respectively. The reverse reaction (i.e., cis to trans isomerization) could be achieved after thermal or visible light activation. The analysis of the theoretically determined equilibrium structure of the transition-state connecting cis and trans isomers on the reaction path indicated that the trans-cis interconversion is pursued via the flipping of the substituent, rather than its rotation around the N=N bond. The kinetics of thermal back-reaction and the effect of the presence of the selected ions on the half-life of the cis form were also investigated and discussed. In the case of L1, the presence of fluoride ions sped the thermal relaxation up, whereas the half-life time of cis-L2 was extended in the presence of tested ions.

Electrochemical simulation of metabolic reduction and conjugation reactions of unsymmetrical bisacridine antitumor agents, C-2028 and C-2053.

Electrochemistry (EC) coupled with analysis techniques such as liquid chromatography (LC) and mass spectrometry (MS) has been developed as a powerful tool for drug metabolism simulation. The application of EC in metabolic studies is particularly favourable due to the low matrix contribution compared to in vitro or in vivo biological models. In this paper, the EC(/LC)/MS system was applied to simulate phase I metabolism of the representative two unsymmetrical bisacridines (UAs), named C-2028 and C-2053, which contain nitroaromatic group susceptible to reductive transformations. UAs are a novel potent class of antitumor agents of extraordinary structures that may be useful in the treatment of difficult for therapy human solid tumors such as breast, colon, prostate, and pancreatic tumors. It is considered that the biological action of these compounds may be due to the redox properties of the nitroaromatic group. At first, the relevant conditions for the electrochemical conversion and product identification process, including the electrode potential range, electrolyte composition, and working electrode material, were optimized with the application of 1-nitroacridine as a model compound. Electrochemical simulation of C-2028 and C-2053 reductive metabolism resulted in the generation of six and five products, respectively. The formation of hydroxylamine m/z [M+H-14]+, amine m/z [M+H-30]+, and novel N-oxide m/z [M+H-18]+ species from UAs was demonstrated. Furthermore, both studied compounds were shown to be stable, retaining their dimeric forms, during electrochemical experiments. The electrochemical method also indicated the susceptibility of C-2028 to phase II metabolic reactions. The respective glutathione and dithiothreitol adducts of C-2028 were identified as ions at m/z 873 and m/z 720. In conclusion, the electrochemical reductive transformations of antitumor UAs allowed for the synthesis of new reactive intermediate forms permitting the study of their interactions with biologically crucial molecules.

Antifungal Activity of Capridine ß as a Consequence of Its Biotransformation into Metabolite Affecting Yeast Topoisomerase II Activity.

In the last few years, increasing importance is attached to problems caused by fungal pathogens. Current methods of preventing fungal infections remain unsatisfactory. There are several antifungal compounds which are highly effective in some cases, however, they have limitations in usage: Nephrotoxicity and other adverse effects. In addition, the frequent use of available fungistatic drugs promotes drug resistance. Therefore, there is an urgent need for the development of a novel antifungal drug with a different mechanism of action, blocking of the fungal DNA topoisomerases activity appear to be a promising idea. According to previous studies on the m-AMSA moderate inhibitory effect on fungal topoisomerase II, we have decided to study Capridine ß (also acridine derivative) antifungal activity, as well as its inhibitory potential on yeast topoisomerase II (yTOPOII). Results indicated that Capridine ß antifungal activity depends on the kind of strains analyzed (MICs range 0.5-64 µg mL-1) and is related to its biotransformation in the cells. An investigation of metabolite formation, identified as Capridine ß reduction product (IE1) by the fungus Candida albicans was performed. IE1 exhibited no activity against fungal cells due to an inability to enter the cells. Although no antifungal activity was observed, in contrast to Capridine ß, biotransformation metabolite totally inhibited the yTOPOII-mediated relaxation at concentrations lower than detected for m-AMSA. The closely related Capridine ß only slightly diminished the catalytic activity of yTOPOII.

A new 1-nitro-9-aminoacridine derivative targeting yeast topoisomerase II able to overcome fluconazole-resistance.

Fungal resistance remains a significant threat and a leading cause of death worldwide. Thus, overcoming microbial infections have again become a serious clinical problem. Although acridine derivatives are widely analyzed as anticancer agents, only a few reports have demonstrated their antifungal activity. In an effort to develop biologically active antifungals, twelve novel C-857 (9-(2'-hydroxyethylamino)-1-nitroacridine) and C-1748 (9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine) derivatives were synthesized. The evaluation of biological properties suggests that starting compounds: C-1748, C-857 and IE3 (2-[(4-methyl-1-nitroacridin-9-yl)amino]ethyl lysinate), IE4 (2-[(1-nitroacridin-9-yl)amino]ethyl lysinate) antifungal mode of action differ from that determined for IE5 (N'-{3-[(4-methyl-1-nitroacridin-9-yl)amino]propyl}lysinamide), IE6 (N'-{3-[(1-nitroacridin-9-yl)amino]propyl}lysinamide) and IE10 (3,3'-Bis-(1-nitroacridin-9-ylamino)-aminoethylaminoethylaminoethylamine). Although MIC values determined for the latter were higher, in contrast to C-857 and C-1748, newly synthesized IE5, IE6 and IE10 reduced C. albicans hyphal growth in different inducing media. Those compounds also exhibited antibiofilm activity, whereas IE10 was the most effective. Moreover, only IE6 exhibited antifungal activity against fluconazole resistant C. albicans strains with MICs values in the range of 16-64 µg mL-1. Our results also indicate that, in contrast to other analyzed derivatives, novel synthetized compounds IE6 and IE10 with antifungal activity target yeast topoisomerase II activity.

Design, synthesis and high antitumor potential of new unsymmetrical bisacridine derivatives towards human solid tumors, specifically pancreatic cancers and their unique ability to stabilize DNA G-quadruplexes.

New promising unsymmetrical bisacridine derivatives (UAs), have been developed. Three groups including 36 compounds were synthesized by the condensation of 4-nitro or 4-methylacridinone, imidazoacridinone and triazoloacridinone derivatives with 1-nitroacridine compounds linked with an aminoalkyl chain. Cytotoxicity screening revealed the high potency of these compounds against several tumor cell lines. Particularly, imidazoacridinone-1-nitroacridine dimers strongly inhibited pancreatic Panc-1, Mia-Pa-Ca-2, Capan-2 and prostate cancer DU-145 cell growth. The studied compounds showed very strong antitumor activity (T/C> 300%) against Walker 256 rat adenocarcinoma. The selected 26 UAs were tested against 12 human tumor xenografts in nude mice, including colon, breast, prostate and pancreatic cancers. The studies on the molecular mechanism of action demonstrated that these unsymmetrical dimers significantly responded to the presence of G-quadruplex not to dsDNA. Structure-activity relationships for UAs potency to G-quadruplex stabilization indicated that thermal stability of this drug-G-quadruplex complex depended not only on the structure of heterocyclic rings, but also on the properties of dialkylamino chains of the ring linkers. In conclusion, the presented studies identified the new group of effective antitumor agents against solid human tumors, particularly pancreatic Panc-1, BxPC-3 and Mia-Pa-Ca-2 and strongly indicated their distinctive interactions with DNA. In contrast to monomers, G-quadruplex not dsDNA is proposed to be the first molecular target for these compounds.

The overexpression of CPR and P450 3A4 in pancreatic cancer cells changes the metabolic profile and increases the cytotoxicity and pro-apoptotic activity of acridine antitumor agent, C-1748.

Drug resistance is one of the major causes of pancreatic cancer treatment failure. Thus, it is still imperative to develop new active compounds and novel approach to improve drug efficacy. Here we present 9-amino-1-nitroacridine antitumor agent, C-1748, developed in our laboratory, as a candidate for pancreatic cancer treatment. We examined (i) the cellular response of pancreatic cancer cell lines: Panc-1, MiaPaCa-2, BxPC-3 and AsPC-1, differing in expression levels of commonly mutated genes for this cancer type, to C-1748 treatment and (ii) the role of P450 3A4 isoenzyme and cytochrome P450 reductase (CPR) in the modulation of this response. C-1748 exhibited the highest cytotoxic activity against MiaPaCa-2, while AsPC-1 cells were the most resistant (IC50: 0.015, 0.075µM, respectively). A considerable amount of apoptosis was detected in Panc-1 and MiaPaCa-2 cells but only limited apoptosis was observed in AsPC-1 and BxPC-3 cells as indicated by morphological changes and biochemical markers. Furthermore, only AsPC-1 cells underwent senescence. Since AsPC-1 cells were the most resistant to C-1748 as evidenced by the lowest P450 3A4 and CPR protein levels, this cell line was subjected to transient transfection either with P450 3A4 or CPR gene. The overexpression of P450 3A4 or CPR changed the pro-apoptotic activity of C-1748 and sensitized AsPC-1 cells to this drug compared to wild-type cells. However, metabolism was changed significantly only for CPR overexpressing cells. In conclusion, the antitumor effectiveness of C-1748 would be improved by multi-drug therapy with chemotherapeutics, that are able to induce P450 3A4 and/or CPR gene expression.