Identification of nonplanar small molecule for G-quadruplex grooves: molecular docking and molecular dynamic study

DNA G-quadruplex is an attractive drug target for anticancer therapy. Most G-quadruplex ligands have little selectivity, due to π-stacking interaction with common G-tetrads surface. Thanks to the varieties of G-quadruplex grooves, the groove-binding ligand is expected to create high selectivity. Therefore, developing novel molecular geometries that target G-quadruplex groove has been paid growing attention. In this work, steroid FG, a special nonplanar and nonaromatic small molecule, interacting with different conformations of G-quadruplexes has been studied by molecular docking and molecular dynamics simulations. The results showed the selectivity of the hydrophobic group of steroid FG for the wide groove of antiparallel G-quadruplex. The methyl groups on the tetracyclic ring of steroid represent the specific binding ability for the small hydrophobic cavity formed by reversed stacking of G-tetrads in antiparallel G-quadruplex groove. This work provides new insight for developing new classes of G-quadruplex groove-binding ligands.     

Specific binding of telomeric G-quadruplexes by hydrosoluble perylene derivatives inhibits repeat addition processivity of human telomerase

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC₅₀ values of HPDIs were in the low micromolar (0.5–5 μM) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase.     

A dual-site simultaneous binding mode in the interaction between parallel-stranded G-quadruplex [d(TGGGGT)]4 and cyanine dye 2,2′-diethyl-9-methyl-selenacarbocyanine bromide

G-quadruplexes have attracted growing attention as a potential cancer-associated target for both treatment and detection in recent years. For detection purpose, high specificity is one of the most important factors to be considered in G-quadruplex probe design. It is well known that end stacking and groove binding are two dominated quadruplex-ligand binding modes, and currently most reported G-quadruplex probes are designed based on the former, which has been proven to show good selectivity between quadruplexes and non-quadruplexes. Because groove of G-quadruplex also has some unique chemical properties, it could be inferred that probes that can interact with both the groove and G-tetrad site of certain G-quadruplexes simultaneously might possess higher specificity in aspects of discriminating different quadruplexes. In this article, we report a cyanine dye as a potential novel probe scaffold that could occupy both the 5′-end external G-tetrad and the corresponding groove of the G-quadruplex simultaneously. By using various spectrum and nuclear magnetic resonance techniques, we give a detailed binding characterization for this dual-site simultaneous binding mode. A preliminary result suggests that this mode might provide highly specific recognition to a parallel-stranded G-quadruplex. These findings and the structural elucidation might give some clues in aspects of developing highly specific G-quadruplex probes.     

Stabilizing parallel G-quadruplex DNA by a new class of ligands: Two non-planar alkaloids through interaction in lateral grooves

Human DNA sequences consisting of tandem guanine (G) nucleotides can fold into a four-stranded structure named G-quadruplex via Hoogsteen hydrogen bonding. As the sequences forming G-quadruplex exist in essential regions of eukaryotic chromosomes and are involved in many important biological processes, the study of their biological functions has currently become a hotspot. Compounds selectively binding and stabilizing G-quadruplex structures have the potential to inhibit telomerase activity or alter oncogene expression levels and thus may act as antitumor agents. Most of reported G-quadruplex ligands generally have planar structures which stabilize G-quadruplex by π–π stacking. However, based on a pharmacophore-based virtual screening two non-planar G-quadruplex ligands were found. These two ligands exhibit good capability for G-quadruplex stabilization and prefer binding to paralleled G-quadruplex rather than to duplex DNA. The binding of these ligands to G-quadruplex may result from groove binding at a 2:1 stoichiometry. These results have shown that planar structures are not essential for G-quadruplex stabilizers, which may represent a new class of G-quadruplex-targeted agents as potential antitumor drugs.     

Pharmacophore-based discovery of triaryl-substituted imidazole as new telomeric G-quadruplex ligand

Discovery of potent and selective ligands for telomeric G-quadruplex DNA is a challenging work. Through a combination approach of pharmacophore model construction, model validation, database virtual screening, chemical synthesis and interaction evaluation, we discovered and confirmed triaryl-substituted imidazole TSIZ01 to be a new telomeric G-quadruplex ligand with potent binding and stabilizing activity to G-quadruplex DNA, as well as a 8.7-fold selectivity towards telomeric G-quadruplex DNA over duplex DNA.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

Structure and Stability of Human Telomeric G-Quadruplex with Preclinical 9-Amino Acridines

G-quadruplexes are higher-order DNA structures formed from guanine-rich sequences, and have been identified as attractive anticancer drug targets. Elucidating the three-dimensional structure of G-quadruplex with 9-amino acridines and the specific interactions involved in binding selectivity are the key to understanding their mechanism of action. Fluorescence titration assays, competitive dialysis and NMR studies have been used to study the binding specificity of 9-amino acridines to DNA. Structural models of the complexes with the telomeric DNA G-quadruplex based on NMR measurements were developed and further examined by molecular dynamics simulations and free energy calculations. Selective binding of 9-amino acridines for G-quadruplex sequences were observed. These compounds bind between A and G-tetrads, involving significant π-π interactions and several strong hydrogen bonds. The specific interactions between different moieties of the 9-amino acridines to the DNA were examined and shown to play a significant role in governing the overall stabilities of DNA G-quadruplex complexes. Both 9-amino acridines, with similar binding affinities to the G-quadruplex, were shown to induce different level of structural stabilization through intercalation. This unique property of altering structural stability is likely a contributing factor for affecting telomerase function and, subsequently, the observed differences in the anticancer activities between the two 9-amino acridines.     

Molecular dynamics and principal components of potassium binding with human telomeric intra-molecular G-quadruplex

Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting telomerase maintenance of telomeres in cancer. Metal cations have been recognized as playing important roles in stabilizing G-quadruplex, but their binding processes to human telomeric G-quadruplex remain uncharacterized. To investigate the detailed binding procedures, molecular dynamics simulations were conducted on the hybrid [3+ 1] form-one human telomeric intra-molecular G-quadruplex. We show here that the binding of a potassium ion to a G-tetrad core is mediated by two alternative pathways. Principal component analysis illustrated the dominant concerted motions of G-quadruplex occurred at the loop domains. MM-PBSA calculations revealed that binding was energetically favorable and driven by the electrostatic interactions. The lower binding site was found more constructive favorable for binding. Our data provide useful information on a potassium-mediated stable structure of human telomeric intra-molecular G-quadruplex, implicating in ion disorder associated conformational changes and targeted drug design.     

Synthesis and evaluation of cationic phthalocyanine derivatives as potential inhibitors of telomerase

A series of water-soluble cationic phthalocyanine derivatives (1-10) were designed and synthesized to develop novel and potent telomerase inhibitors. These phthalocyanine derivatives as inhibitors of telomerase were investigated via modified telomerase repeat amplification protocol (TRAP) assay. The TRAP assay indicates that these cationic compounds had strong telomerase inhibitory activity (IC(50)<1.65 microM). To determine whether the phthalocyanine derivatives binding to G-quadruplex enhance the block to DNA synthesis, primer extension reactions were carried out in the presence of phthalocyanines. The interaction of the G-quadruplex of telomerase DNA with these molecules was examined by CD melting and PCR stop assay. These cationic phthalocyanine derivatives can stabilize G-quadruplex, which is demonstrated by the increased T(m) values. All these results indicate that the phthalocyanine derivatives might be potential lead compounds for the development of new telomerase inhibitor.     

Amide bond direction modulates G-quadruplex recognition and telomerase inhibition by 2,6 and 2,7 bis-substituted anthracenedione derivatives

G-quadruplex structures of DNA represent a potentially useful target for anticancer drugs. Stabilisation of this arrangement at the ends of chromosomes may inhibit the action of telomerase, an enzyme involved in immortalization of cancer cells. Appropriately substituted amido anthracenediones are effective G-quadruplex stabilizers, but no information is available as yet on the possible modulation of G-quadruplex recognition and telomerase inhibition produced by the direction of the amide bond. To understand the basis of amido anthracenedione selectivity, we have synthesized a number of derivatives bearing the -CO-NH- or -NH-CO- group linked to the planar anthraquinone (AQ) moiety at 2,6 and 2,7 positions. The various isomers were tested in terms of telomerase inhibition, determined by the TRAP assay, G-quadruplex stabilisation measured by the increase in melting temperature of the appropriately folded oligonucleotide using FRET, and conformational and G4 binding properties examined by molecular modelling techniques. In all cases, enzymatic inhibition and G-quadruplex stabilization were directly related, which strongly supports the proposed molecular mechanism of telomerase interference. Interestingly, the AQ-NH-CO- arrangement performs invariantly better than the AQ-CO-NH- arrangement, showing a clear preference among isomeric derivatives. Theoretical calculations suggest that the former amide arrangement is co-planar with the aromatic system, whereas the latter is tilted by about 30 degrees when considering the most stable conformation. A more extended planar surface would allow more efficient stacking interactions with the quadruplex structure, hence more effective telomerase inhibition.     

The relationship between ligand aggregation and G-quadruplex DNA selectivity in a series of 3,4,9,10-perylenetetracarboxylic acid diimides

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.     

Tri-, tetra- and heptacyclic perylene analogues as new potential antineoplastic agents based on DNA telomerase inhibition

A recent approach in anticancer chemotherapy envisages telomerase as a potentially useful target. An attractive strategy deals with the development of compounds able to stabilize telomeric DNA in the G-quadruplex folded structure and, among them, a prominent position is found in the perylenes. With the aim to further investigate the role of drug structure, in view of possible pharmaceutical applications, we synthesized a series of compounds related to PIPER, a well-known perylene-based telomerase inhibitor. We modified the number of condensed aromatic rings and introduced different side chains to modulate drug protonation state and extent of self-aggregation. Effective telomerase inhibition was induced by heptacyclic analogues only, some showing a remarkably wide selectivity index with reference to inhibition of Taq polymerase. G-quadruplex stabilization was monitored by circular dichroism and melting experiments. Cell cytotoxicity measurements indicated a poor short-term cell killing ability for the best G-quartet binders. Besides the presence of a planar seven-condensed ring system, the introduction of a cyclic amine in the side chains critically affects the selectivity window.     

Natural and synthetic G-quadruplex interactive berberine derivatives

The interaction of the natural alkaloid berberine with various G-quadruplex DNA structures and its ability to inhibit telomerase have been examined and compared with those of a synthetic piperidino derivative and the related compound coralyne. The results show that these molecules have selectivity for G-quadruplex compared to duplex DNA, and that their aromatic moieties play a dominant role in quadruplex binding.     

Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC(50) values of ～0.1 μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.     

A DNA polymerase stop assay for G-quadruplex-interactive compounds.

We have developed and characterized an assay for G-quadruplex-interactive compounds that makes use of the fact that G-rich DNA templates present obstacles to DNA synthesis by DNA polymerases. Using Taq DNA polymerase and the G-quadruplex binding 2, 6-diamidoanthraquinone BSU-1051, we find that BSU-1051 leads to enhanced arrest of DNA synthesis in the presence of K+by stabilizing an intramolecular G-quadruplex structure formed by four repeats of either TTGGGG or TTAGGG in the template strand. The data provide additional evidence that BSU-1051 modulates telomerase activity by stabilization of telomeric G-quadruplex DNA and point to a polymerase arrest assay as a sensitive method for screening for G-quadruplex-interactive agents with potential clinical utility.     

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC₅₀ values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.