Specific stabilization of promoter G-Quadruplex DNA by 2,6-disubstituted amidoanthracene-9,10-dione based dimeric distamycin analogues and their selective cancer cell cytotoxicity

doi:10.1016/j.ejmech.2020.112202

European Journal of Medicinal Chemistry

Volume 195, 1 June 2020, 112202

https://doi.org/10.1016/j.ejmech.2020.112202 Get rights and content

Highlights

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Oligopyrrole carboxamides conjugated anthraquinone derivatives stabilize G-quadruplex DNA over duplex DNA.
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Oligopyrrole carboxamides conjugated anthraquinone derivatives stabilize promoter G-quadruplex DNA.
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Stacking mode of interaction of the ligand with c-MYC G-quadruplex DNA.
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Selective cancer cell cytotoxicity over normal cells was obtained upon treatment of these ligands.
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Cellular morphological changes and nuclear condensation observed in the ligand treated cells.

Abstract

We have designed and synthesized anthraquinone containing compounds which have oligopyrrole side chains of varying lengths. These compounds stabilized the G-quadruplex DNA formed in the promoter regions of c-MYC oncogenes selectively over the duplex DNA. These observations were recorded using UV–vis spectroscopic titrations, fluorescence measurements and circular dichroism (CD) spectral titrations. The potency of the compounds to stabilize the G4 DNA has been shown from the thermal denaturation experiments. The compound interacts with c-MYC G-quadruplex DNA through stacking mode as obtained from ethidium bromide displacement assay, cyclic voltammetric titration, and docking experiments. Molecular modeling studies suggested that the stacking of the anthraquinone moiety over the G-tetrad of the G4 structures are responsible for the stability of such quadruplex secondary structure. Furthermore, polymerase stop assay also supported the formation of stable G4 structures in the presence of the above-mentioned compounds. The compounds have shown selective cancer cell (HeLa and HEK293T) cytotoxicity over normal cells (NIH3T3 and HDFa) under in vitro conditions as determined from MTT based cell viability assay. Apoptosis was found to be the mechanistic pathway underlying the cancer cell cytotoxicity as obtained from Annexin V-FITC and PI dual staining assay which was further substantiated by nuclear morphological changes as observed by AO/EB dual staining assay. Cellular morphological changes, as well as nuclear condensation and fragmentation upon treatment with these compounds, were observed under bright field and confocal microscopy.

Graphical abstract

Introduction

Guanine-rich short deoxyribonucleic acid sequences can fold into G-quadruplex (G4) DNA structures arising out of various non-covalent supramolecular interactions such as Hoogsteen type hydrogen bonds (between N₁–O₆ and N₂–N₇) and π–π stacking interactions among the heteroatomic nucleobases [1]. The central cavities of the four-stranded G4 assembly are further stabilized by monovalent cations such as Na⁺ and K⁺ which coordinate with the O₆ lone pair of each guanine moiety, which in turn reduces the charge repulsion [2]. G-quadruplex DNA structures adopt various topologies such as parallel, anti-parallel, mixed hybrid etc. depending upon the length of the G-rich sequences, size/length of the loop, the conformation of the glycosidic bonds, and the presence of the centrally coordinated metal ions [1,3]. Intermediate sequences between the G-tetrad forming guanines form loops which play a significant role in regulating the nature of folding and stability of the G-quadruplex [4]. Further, depending upon the number of participating strands in the quadruplex DNA structure, G-quadruplexes can be classified into three groups such as tetramolecular, bimolecular, and monomolecular, or intramolecular [5]. Such G-quadruplex DNA structures have indeed been detected and visualized at the cellular level by employing a highly structure-specific antibody [6].

Various cellular processes like transcription, translation, the activity of telomerase enzyme may be regulated by stabilizing the G-quadruplex DNA structures that have been recognized in the human genome such as in the promoter region of some oncogenes as well as in the telomeric 3′-overhangs that comprise a tandem repeat of the d(TTAGGG)n units [7]. The promoter regions of many proto-oncogenes like c-MYC, c-KIT, BCL-2, k-RAS, VEGF, HIF-1α contain G-rich sequences that have a high tendency of forming G4 structures [8]. Thus, small molecules, which can stabilize these G-quadruplex DNA structures, may also downregulate the protein expressions and inhibit the cellular functions that can eventually lead to cell death [9]. In a general strategy, small molecules which have large π-surface(s) are designed to interact with the G-tetrad through π-π interactions and neutral/cationic side chains are incorporated to favorably interact with the phosphate backbone of the groove/loop to get adequate quadruplex binding selectivity over duplex DNA [10].

Anthraquinone-based compounds have shown effective DNA binding properties and can act as anti-cancer agents [11]. Anthraquinone is the structural element of anthracyclines like doxorubicin and daunomycin which have been introduced in clinical uses for the treatment of various types of cancers and are well-known DNA binders [12]. Besides these, anthraquinone derivatives have various pharmacological applications like anti-inflammatory, antiarthritic, antifungal, antibacterial and moreover, they have shown potential activity in malaria treatment and multiple sclerosis [13].

In the last few decades, many chemical compounds have been synthesized and studied for the selective stabilization of G-quadruplex DNA [14]. A major challenge for designing G-quadruplex mediated anti-cancer agents is to improve the selectivity for binding towards G-quadruplex over duplex DNA [15]. Small molecules containing acridine, furan, salen, anthraquinone, carbazole, benzimidazole, salphen, xanthone pharmacophore have been extensively studied for the stabilization of G-quadruplex DNA [16]. Anthraquinones were one of the first reported ligands that have shown affinity towards G-quadruplex structures as well as inhibited telomerase activity [17]. The anthraquinone derivatives have received much attention due to their large <pi>-surface which can efficiently do π-π interaction with the G-tetrads. In 1997, Neidle and Hurley have first reported a 2,6-diaminoalkylamidoanthraquinone derivative, BSU-1051 as the first G-quadruplex ligand and telomerase inhibitor [17]. Subsequently, the disubstituted amido-anthraquinone derivatives are further developed at different substituent positions with varying lengths of side chains to get binding selectivity towards G-quadruplex DNA [18]. Anthraquinone moieties have been conjugated with amino acids and amino sugars to regulate their G4 DNA binding affinities [19]. Moreover, Huang et al. reported several disubstituted amido and aminoanthraquinone derivatives as human telomerase inhibitors and potential anti-cancer agents [20]. Interestingly, different chemically modified heteroarene fused anthraquinone derivatives have been explored by Shchekotikhin and co-workers as human telomeric G-quadruplex stabilizers and antiproliferative agents [21]. However, owing to their diverse medicinal significances, there is a sustained and continuing interest in search of new anthraquinone derivatives to improve their biological activities.

Naturally occurring oligopeptides like netropsin, distamycin, anthelvencin, norformycin, etc. have attracted the synthetic chemists as well as molecular biologists because of their various biological significances such as antiviral, antibacterial and anti-cancer activities [22]. Oligopyrroles such as netropsin and distamycin A are one of the most well-studied polyamides that bind in the minor groove of duplex DNA with AT-rich sequence [23]. The binding affinities of these oligopyrroles were acquired from H-bonding interactions with bases, electrostatic interactions with the phosphate backbones and van der Waals contacts with the minor groove [24]. These oligopyrroles are also well-known for the binding to the grooves of the G-quadruplex DNA, but the selectivity is comparatively poor [25]. However, we have reported earlier that when the oligopyrroles are conjugated with salen and salphen moieties, they have shown promising selectivity towards G-quadruplex DNA over duplex DNA [26]. To a similar manner, we propose that the conjugation of the oligopyrrole carboxamides with anthraquinone pharmacophore, the selectivity of the compounds towards stabilization of G-quadruplex DNA may be obtained efficiently. The rationale behind the design of the molecules presented herein is to focus on the dual recognition ability as the stacking interaction of the anthraquinone moiety to the G-tetrad and the interaction with grooves/loops with the flexible side chains. This may increase the probability of achieving selectivity of the G-quadruplex DNA over the ds-DNA.

Herein, we have synthesized new anthraquinone derivatives conjugated with different numbers of oligopyrroles to stabilize G4 structures. The compounds are designed in such a way to address some important parameters like molecular length, flexibility. The interaction of these compounds with promoter c-MYC, c-KIT1, c-KIT2 G4, and telomeric G4 DNA were studied by UV–vis spectroscopic titration. Also, the selectivity for the binding of the compounds towards G4 DNA over duplex DNA was determined from this experiment. The stabilization of G-quadruplex DNA by these compounds was also supported by fluorescence titration experiment along with CD titration, CD melting experiment, Taq polymerase stop assay. To determine the mode of interactions of these compounds with G4 DNA, ethidium bromide displacement assay, cyclic voltammetric titration, and molecular modeling studies were carried out. Further, to check the potency of these compounds in vitro condition, we have performed the MTT based cell viability assay in HeLa (human cervical cancer transformed cells), HEK 293T (human embryonic kidney transformed cells) over normal cells like NIH3T3 (primary mouse embryonic fibroblast cells) and HDFa (Human Dermal Fibroblast, adult). To ascertain the mechanistic pathway of cell death, Annexin V and PI dual staining assay were employed which further has been verified by fluorescence microscopy with AO/EB staining method. Morphological changes of the cells and nuclear condensation upon treatment with these compounds were followed by bright field microscopy and confocal microscopy respectively.

Section snippets

Results and discussion

Design and Synthesis. We have synthesized mono-, di- and tri-pyrrole compounds and have attached the varying lengths of flanking side chains to the anthraquinone moiety (Fig. 1). The side chains contain tetramethylammonium groups which are further conjugated with oligopyrrole carboxamides of different lengths to observe the role in the structure-activity relationship. The molecular length, as well as the shape of the ligands, were varied by varying the number of the pyrrole rings attached to

Conclusions

Anthraquinone derivatives are already known for the inhibition of telomerase enzyme [17] and oligopyrrole moieties are reported as groove binders for duplex DNA [23]. However, the conjugation of the oligopyrrole carboxamide with the anthraquinone chromophore is not reported so far as a target for the stabilization of G4 DNA. Hence, we have designed and synthesized disubstituted anthraquinone derivatives tagged with oligopyrrole moieties which have shown significant interactions with different

Experimental section

Materials. All the starting materials and solvents were purchased from the best known available commercial sources and used as it is without any further purification. Final compounds were thoroughly analyzed by ¹H NMR, ¹³C NMR, mass spectra, FT-IR, and elemental analysis. ¹H and ¹³C NMR spectra were recorded on Bruker Avance 400 and 800 (400 MHz for ¹H; 100 and 200 (with cryoprobe) MHz for ¹³C) spectrometer. Mass and IR spectra were recorded on Micromass Q-TOF Micro TM spectrometer and FT-IR

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by a J. C. Bose Fellowship, Department of Science and Technology (DST) to SB. SR thanks the Council of Scientific and Industrial Research (CSIR) for a senior research fellowship. We would like to acknowledge the central confocal and flow cytometry facilities, Division of Biological Sciences, IISc, for the confocal imaging and FACS.

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Spectroscopy evidence of interaction of 1,5 disubstituted piperidino-amido anthraquinone derivative with human telomeric G-quadruplex DNA: Basis of anticancer action
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The binding of ligands to G-quadruplex (G4) structures at the ends of human telomeric DNA induces thermal stabilization, which interferes with telomere maintenance by disrupting the association of telomeres with the telomerase enzyme—an important marker for cancer. Understanding the binding mode of the ligand-G quadruplex DNA complex is imperative for evaluating the relative efficacy and specificity of their interaction. We focused on the interaction of 1,5-Bis(3-piperidino propionamido) anthracene-9,10-dione with human telomeric DNA sequences using surface plasmon resonance, absorbance, fluorescence (steady-state and lifetime), and circular dichroism spectroscopy techniques. The ligand binding with HTel22 (K_b = 8.4 × 10⁵ M⁻¹) induced cell toxicity with an IC₅₀ value of ∼ 8.64 µM in MCF-7 cancer cell lines. Significant hypochromism (59 %), fluorescence quenching (97 %), no change in fluorescence lifetime and absence of induced Circular Dichroism (CD) band upon addition of G4 DNA to ligand, suggest a groove/external binding mode. CD spectral changes reflect rearrangement in parallel and antiparallel strands to accommodate the ligand. Docking results reveal specific short contacts of the ligand's side chain, including 9CO, 13N, 14NH, and 11CO, with the grooves of the G4 DNA, without making any contact with the loops, favoring an energetically more favorable conformation. Thermal denaturation profiles, obtained by Differential Scanning Calorimetry and CD, show the stabilization of wHTel26 and HTel22 G4 DNA in K⁺ and Na⁺ rich solutions by 21.2 and 17.6 °C, respectively, which may restrict the access of telomerase to telomeres. The findings indicate the potential of modifying anthraquinone substituent groups for therapeutic applications.
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G-quadruplex is a non-canonical secondary structure identified in the telomeric region and the promoter of many oncogenes. Anthraquinone derivatives, a well-known inducer of telomere disruption in malignant cells and activate the apoptotic pathway. We used biophysical and biochemical studies to confirm the interaction of synthesized anthraquinone derivatives with the human telomeric G-quadruplex sequence. The binding affinity of N-2DEA and N-1DEA are K_b = 4.8 × 10⁶ M⁻¹ and K_b = 7.6 × 10⁵ M⁻¹, respectively, leading to hypochroism, fluorescence quenching with minor redshift and ellipticity variations indicating ligand binding in the external groove. We found that sodium ions induced stabilization more rather than potassium ions. Molecular docking of complex demonstrates a molecule’s exterior binding to a quadruplex. The investigation of ROS activity indicated that the cell initiates mortality in response to the IC₅₀ concentration. Cellular morphology, nuclear condensation, and fragmentation were altered in the treated cell, impairing cellular function. Finally, the transcriptional regulatory study paves the way for drug design as an anti-cancer agent because of the tremendous possibilities of changing substituent groups on anthraquinones to improve efficacy and selectivity for G-quartet DNA. Our research focused on how ligand binding to telomere sequences induces oxidative stress and inhibits the growth of malignant cells.
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The dihydroindolizino indole derivative comprising the 4-cyanophenyl group could bind efficiently to G-quadruplex formed in the telomeric region and NHE III1, a c-MYC promoter region [115]. The anthraquinone compounds having a varying length of side chains could stabilize the G-quadruplex DNA (via stacking mode) in the promoter region of the c-MYC oncogene in comparison to duplex DNA [94]. Wu et al. have synthesized an imidazole-benzothiazole conjugate (IZTZ-1) that could favourably stabilize the G-quadruplex DNA present in the promoter region of the c-MYC gene [95].
Guanine-rich DNA sequences may fold back into non-canonical four-stranded secondary structures termed as G-quadruplexes. The role of G-quadruplexes has already been well established in different diseases like cancer, neurological and viral disorders etc. Also, several small molecules have been reported, which can influence the involvement of G-quadruplexes either through stabilization or destabilization in the cellular environment. Growing statistics have associated G-quadruplex assemblies to a discrete biological process in vivo, including DNA replication, transcription, genomic stability, and epigenetic regulation. DNA G-quadruplex existence in human telomere is well recognized attractive target for anticancer drugs. G-quadruplex-interactive ligands have been known to prevent telomerase access as well as telomere capping. To the best of our understanding, the role of G-quadruplexes in virology, neuropharmacology, cancer progression and its treatment has not been discussed on a single platform till date. This review aims to enhance our knowledge regarding these magical sticky quadruplex structures, which have been quite significantly proved to be the part of many cellular processes along with their established in vivo existence. Understanding regarding stabilizing or destabilizing ligands of these multistranded guanine quadruplex structures might be proved as the facilitator of drug discovery process for many incurable diseases in future.
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Furthermore, the effect of 2a and 2b on the conformation of the promoter and mitochondrial G-quadruplexes was investigated in 10 mM Tris-HCl buffer containing 100 mM KCl at pH 7.0 by Circular Dichroism (CD) spectroscopy (Fig. S9). According to the characteristic peaks of CD spectra, bcl-2 [40] and HRCC [41] exhibit a mixed-type conformation, both c-kit 2 [42] and c-myc [17] are a parallel conformation, while KSS is the typical of hybrid conformation [41]. With the addition of the compounds, CD spectra of G-quadruplexes are no obvious change, suggesting that both 2a and 2b fail to induce their conformation transformation.
G-quadruplex DNA has attracted the widespread attention as a novel target of anticancer strategy. Herein, two novel stilbene derivatives 2a and 2b were designed and synthesized under mild reaction conditions, and their interactions with G-quadruplex DNA, cytotoxicity, and distribution in living cells were investigated in detail. Both compounds display a low cytotoxicity and the higher affinity to G-quadruplex DNA than to the other secondary structures, including duplex, single-stranded and i-motif DNA, moreover, the affinity of 2b with m-allyl pyridine salt group to G-quadruplex DNA is about 10-fold stronger than that of 2a with p-allyl pyridine salt group. The interactions of the compounds with the promoter G-quadruplexes are enthalpy-driven by an ITC assay. 2a and 2b not only stabilize the G-quadruplex structure but also induce the G-rich sequences (bcl-2, HRCC and KSS) to fold into the mixed-type G-quadruplex in Na⁺/K⁺ free Tris-HCl buffer at pH 7.0, and 2b presents the higher stabilization to G-quadruplex than 2a by a FRET-melting assay. 2b presents a dual-emission at 508 and 600 nm and gives a turn-on and stronger and more sensitive fluorescence response over 2a to the promoter (bcl-2, c-kit 2 and c-myc) and mitochondrial (HRCC and KSS) G-quadruplex DNA at both emission wavelengths, moreover, the peak at 508 nm is blue-shifted to 466 nm after binding to DNA. The blue and red dual-channel CLSM images indicate that 2b is mainly distributed in the mitochondrion of living HepG2 cells. The results show that 2b is a potential dual-channel fluorescent probe for mitochondrial G-quadruplex DNA in living cells.
Highly selective turn-on red fluorescence probes for visualization of the G-quadruplexes DNA in living cells
2022, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
Studies on small molecule fluorescent probes for detecting G-quadruplexes DNA have bring about an extensive attention in recent years. In this paper, we designed and synthesized three benzothiazole derivatives named 2a-2c under moderate reaction conditions and investigated their interactions with DNA (single-stranded, duplex, i-motif and G-quadruplex) and distribution in living cell. Three compounds present a large Stokes shift (∼90 nm) and a weak red fluorescence emission, and they exhibit a good selectivity and sensitive turn-on fluorescence response for the promoter G-quadruplex DNA (bcl-2, c-myc and c-kit 2) and mitochondria G-quadruplex (KSS). The affinity of 2a and 2b with N-alkyl side chain group to DNA is stronger than that of 2c with an anion group, therefore, they also increase the stability of the G-quadruplex structure. 2b induces the conformational change of both bcl-2 and KSS G-quadruplexes, while all compounds induce the folding of bcl-2 from the coiled structure to the hybrid G-qrudruplex. Three compounds interact with the G-quadruplex DNA mainly by end-stacking mode. Furthermore, MTT assays and confocal fluorescence images show that these compounds can enter the living HepG2 cells with low cytotoxicity. 2a-2c are mainly located in the mitochondrion and interacted with mitochondria G-quadruplex DNA, while only weak fluorescence can be found in cell nucleus. In a word, 2a-2c can be implied in image of G-quadruplex DNA in living cells.

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Research paperSpecific stabilization of promoter G-Quadruplex DNA by 2,6-disubstituted amidoanthracene-9,10-dione based dimeric distamycin analogues and their selective cancer cell cytotoxicity

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Research paper
Specific stabilization of promoter G-Quadruplex DNA by 2,6-disubstituted amidoanthracene-9,10-dione based dimeric distamycin analogues and their selective cancer cell cytotoxicity

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K⁺: insights into loop interactions of the parallel G-quadruplexes