靶向G-四链体的小分子配体在癌症治疗中应用的研究进展

G-四链体是一类由Hoogsteen氢键维持稳定的,富含鸟嘌呤的DNA或RNA二级结构。人类基因组中存在大量潜在的形成G-四链体的序列,所形成的G-四链体结构能够调控基因组的稳定性、DNA复制和基因表达,其中包括很多与癌症相关基因。因此寻找能够诱导DNA的G富集区域形成G-四链体结构的配体,进而筛选潜在抗癌药物的先导化合物,已成为癌症治疗研究的热点之一。本文对近年来发现和设计的以G-四链体为靶点的小分子配体,按照靶向的G-四链体结构类型和配体的分子结构进行分类,综述了这类化合物在癌症治疗方面的研究进展,分析了相关靶向治疗存在的问题,并对未来的研究方向进行了展望。     

Identification of Compounds that Selectively Stabilize Specific G‐Quadruplex Structures by Using a Thioflavin T‐Displacement Assay as a Tool

Small molecules are used in the G‐quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT‐displacement high‐throughput screening assay to find novel and selective G4‐binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4‐structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.     

Development of a high-throughput G4-FID assay for screening and evaluation of small molecules binding quadruplex nucleic acid structures

G4-FID (G-quadruplex fluorescent intercalator displacement) is a simple and fast method that allows to evaluate the affinity of a compound for G-quadruplex DNA and its selectivity towards duplex DNA. This assay is based on the loss of fluorescence of thiazole orange (TO) upon competitive displacement from DNA by a putative ligand. We describe here the development of a high-throughput version of this assay performed in 96-well microplates, and fully transposable to 384-well microplates. The test was calibrated with a set of G-quadruplex ligands characterized for their ability to bind quadruplex within a large range of affinity. The comparison of the results obtained in microplates and in cuvettes was conducted indicating a full agreement. Additionally, the spectral range of the test was enlarged using two other fluorescent on/off probes whose absorption are red-shifted (TO-PRO-3) and blue-shifted (Hoechst 33258) as compared to that of TO. These labels enable to screen a large diversity of compounds with various optical properties, which was exemplified by evaluation of affinity and selectivity of the porphyrin TMPyP4 that could not be evaluated previously. Altogether, our study demonstrates that the HT-G4-FID assay offers the possibility to label a large variety of G-quadruplexes of biological interest and should enable screening of collections of putative G4-ligands of high structural diversity. It thus represents a powerful tool to bring into light new ligands able to discriminate between quadruplexes of different structures.     

DNA G-四链体识别探针研究进展

G-四链体是一种由富含鸟嘌呤核酸序列形成的独特的二级结构,广泛分布于真核生物基因组,如端粒DNA、r DNA和一系列基因中的启动子区域。G-四链体结构对很多重要的生理过程如基因的转录、复制、重组以及保持染色体的稳定性方面具有重要作用。G-四链体的特异、高灵敏检测将为进一步了解G-四链体结构在人类细胞基因组中的分布、功能和机制奠定基础,也可能为靶向G-四链体的肿瘤治疗方法提供新的思路。因而过去几十年人们一直致力于开发设计具有高选择性和高灵敏度的G-四链体识别探针,这些探针已经广泛应用于溶液中G-四链体的识别,而且具有良好的选择性。目前也有少数探针能够直接用于检测活体G-四链体结构。本文综述了一些常见的靶向G-四链体的小分子配体,以及它们在染色体和活体细胞G-四链体检测中的应用。笔者希冀本文能为设计识别G-四链体的高性能探针,进一步实现活细胞内G-四链体的检测提供借鉴。     

Direct and Single‐Molecule Visualization of the Solution‐State Structures of G‐Hairpin and G‐Triplex Intermediates

We present the direct and single‐molecule visualization of the in‐pathway intermediates of the G‐quadruplex folding that have been inaccessible by any experimental method employed to date. Using DNA origami as a novel tool for the structural control and high‐speed atomic force microscopy (HS‐AFM) for direct visualization, we captured images of the unprecedented solution‐state structures of a tetramolecular antiparallel and (3+1)‐type G‐quadruplex intermediates, such as G‐hairpin and G‐triplex, with nanometer precision. No such structural information was reported previously with any direct or indirect technique, solution or solid‐state, single‐molecule or bulk studies, and at any resolution. Based on our results, we proposed a folding mechanism of these G‐quadruplexes.     

Monitoring G-quadruplex structures and G-quadruplex-ligand complex using 2-aminopurine modified oligonucleotides

The fluorescent base 2-aminopurine (2Ap) was incorporated into the human telomeric DNA sequence d[AGGG(TTAGGG)₃]. The substitution of 2Ap for A in the TTA loops did not affect the G-quadruplex stability. Interestingly, a significant change in the fluorescence intensity of 2Ap between the G-quadruplex and duplex was observed. Therefore, we demonstrated that 2Ap can be used to monitor the duplex to quadruplex conformational change in the human telomeric DNA sequence. This mechanism is explained by the difference in the base stacking in the TTA loop region. Moreover, these probes distinguished between the basket-type and propeller-type G-quadruplexes. We also demonstrated the detection of the telomerase inhibitor agent, such as TMPyP4, using a 2Ap modified telomeric DNA. The formation of the G-quadruplex-ligand complex was observed by the fluorescence titration of TMPyP4.     

Tridentate N-donor palladium(II) complexes as efficient coordinating quadruplex DNA binders

Fifteen complexes of palladium, platinum, and copper, featuring five different N‐donor tridentate (terpyridine‐like) ligands, were prepared with the aim of testing their G‐quadruplex–DNA binding properties. The fluorescence resonance energy transfer melting assay indicated a striking positive effect of palladium on G‐quadruplex DNA stabilization compared with platinum and copper, as well as an influence of the structure of the organic ligand. Putative binding modes (noncoordinative π stacking and base coordination) of palladium and platinum complexes were investigated by ESI‐MS and UV/Vis spectroscopy experiments, which all revealed a greater ability of palladium complexes to coordinate DNA bases. In contrast, platinum compounds tend to predominantly bind to quadruplex DNA in their aqua form by noncoordinative interactions. Remarkably, complexes of [Pd(ttpy)] and [Pd(tMebip)] (ttpy=tolylterpyridine, tMebip=2,2′‐(4‐p‐tolylpyridine‐2,6‐diyl)bis(1‐methyl‐1H‐benzo[d]imidazole)) coordinate efficiently G‐quadruplex structures at room temperature in less than 1 h, and are more efficient than their platinum counterparts for inhibiting the growth of cancer cells. Altogether, these results demonstrate that both the affinity for G‐quadruplex DNA and the binding mode of metal complexes can be modulated by modifying either the metal or the organic ligand.     

Structural basis for telomeric G-quadruplex targeting by naphthalene diimide ligands

The folding of the single-stranded 3' end of the human telomere into G-quadruplex arrangements inhibits the overhang from hybridizing with the RNA template of telomerase and halts telomere maintenance in cancer cells. The ability to thermally stabilize human telomeric DNA as a four-stranded G-quadruplex structure by developing selective small molecule compounds is a therapeutic path to regulating telomerase activity and thereby selectively inhibit cancer cell growth. The development of compounds with the necessary selectivity and affinity to target parallel-stranded G-quadruplex structures has proved particularly challenging to date, relying heavily upon limited structural data. We report here on a structure-based approach to the design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA. Crystal structures have been determined of complexes between a 22-mer intramolecular human telomeric quadruplex and two potent tetra-substituted naphthalene diimide compounds, functionalized with positively charged N-methyl-piperazine side-chains. These compounds promote parallel-stranded quadruplex topology, binding exclusively to the 3' surface of each quadruplex. There are significant differences between the complexes in terms of ligand mobility and in the interactions with quadruplex grooves. One of the two ligands is markedly less mobile in the crystal complex and is more quadruplex-stabilizing, forming multiple electrostatic/hydrogen bond contacts with quadruplex phosphate groups. The data presented here provides a structural rationale for the biophysical (effects on quadruplex thermal stabilization) and biological data (inhibition of proliferation in cancer cell lines and evidence of in vivo antitumor activity) on compounds in this series and, thus, for the concept of telomere targeting with DNA quadruplex-binding small molecules.     

Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G‐Quadruplex

In investigating the binding interactions between the human telomeric RNA (TERRA) G‐quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ‐selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single‐molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force‐jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G‐quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA‐GQ–ligand complex may inspire new strategies for the selective stabilization of G‐quadruplexes in cells.     

Recognizing Hybrid/Mixed G-quadruplex in Human Telomeres by Using a Cyanine Dye Supramolecule with Confocal Laser Scanning Microscopy

Single‐stranded telomeric DNA tends to form a four‐base‐paired planar structure termed G‐quadruplex. Although kinds of G‐quadruplex structures in vitro have been documented in the presence of potassium or sodium, recognition of these DNA motifs (both in vitro and in vivo) is still an important issue in understanding the biological function of the G‐quadruplex structures in telomeres as well as developing anticancer agents. Herein we address this important question through the distinctive properties of a supramolecular system of cyanine dye 3,3′‐di(3‐sulfopropyl)‐4,5,4′,5′‐dibenzo‐9‐methyl‐thiacarbocyanine triethylammonium salt (MTC) upon binding to different DNA motifs. Interaction of MTC with hybrid/mixed G‐quadruplex results in a set of unique spectrophotometric signatures which are completely different from those arising from binding to other DNA motifs. Furthermore, such feature could be extended to map the locations of DNAs on interface. Linear duplex and mixed G‐quadruplex in human telomeres assembled on Au film and stained by MTC were directly recognized by confocal laser scanning microscopy (CLSM). All results suggested that MTC supramolecular system may be a good probe of specific G‐quadruplex structure.     

Templated Formation of Discrete RNA and DNA:RNA Hybrid G‐Quadruplexes and Their Interactions with Targeting Ligands

G‐rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper‐catalyzed azide‐alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G‐quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.     

Dynamics of Fluorescent Dyes Attached to G‐Quadruplex DNA and their Effect on FRET Experiments

FRET spectroscopy is a promising approach for investigating the dynamics of G‐quadruplex DNA folds and improving the targeting of G‐quadruplexes by potential anticancer compounds. To better interpret such experiments, classical and replica‐exchange molecular dynamics simulations and fluorescence‐lifetime measurements are used to understand the behavior of a range of Cy3‐based dyes attached to the 3′ end of G‐quadruplex DNA. The simulations revealed that the dyes interact extensively with the G‐quadruplex. Identification of preferred dye positions relative to the G‐quadruplex in the simulations allows the impact of dye–DNA interactions on FRET results to be determined. All the dyes show significant deviations from the common approximation of being freely rotating and not interacting with the host, but one of the Cy3 dye analogues is slightly closer to this case.     

Study of binding affinity and selectivity of perylene and coronene derivatives towards duplex and quadruplex DNA by ESI‐MS

In this paper, we report an extensive electrospray ionization mass spectrometry (ESI‐MS) study of the noncovalent interactions between different intermolecular and intramolecular G‐quadruplex structures and several perylene and coronene ligands. The selectivity of these compounds toward quadruplex structures with respect to duplex DNA, a fundamental topic for the biological evaluation and the pharmacological application of these ligands as potential chemotherapeutic agents, has also been investigated. After exploring this topic according to the classical approach based on the very simple duplex model of an autocomplementary dodecamer, we extended our analysis reporting for the first time a competition ESI‐MS experiment in the presence of genomic DNA fragments. Whereas those ligands showing a high level of selectivity between quadruplex and duplex oligonucleotides, in terms of binding constants and percentage of bound DNA, confirmed their selectivity in the competition experiment, the contrary was not always true: some ligands showing poor selectivity with the autocomplementary dodecamer resulted selective in the presence of genomic DNA fragments. This result suggests that physiologically nonrelevant interactions are possible with a short duplex oligonucleotide. This means that the dodecamer can fail in representing a biologically significant structural model, or, better, that it can be used to quickly screen potentially selective molecules, but bearing in mind the high probability of false negative results. Copyright © 2008 John Wiley & Sons, Ltd.     

A Light‐up Logic Platform for Selective Recognition of Parallel G‐Quadruplex Structures via Disaggregation‐Induced Emission

The design of turn‐on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G‐quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition‐driven disaggregation (on‐signal) of an ultrabright coumarin‐quinazoline conjugate. The synthesized probe selectively lights‐up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label‐free molecular logic system. Finally, our molecule preferentially stains the G4‐rich nucleoli of cancer cells.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

Formation of Human Telomeric G-quadruplex Structures Induced by the Quaternary Benzophenanthridine Alkaloids:Sanguinarine,Nitidine,and Chelerythrine

The ligands which can facilitate the formation and stabilize G‐quadruplex structures have attracted enormous attention due to their potential ability of inhibiting the telomerase activity and halting tumor cell proliferation. It is noteworthy that the abilities of the quaternary benzophenanthridine alkaloids (QBAs), the very important G‐quadruplex binders, in inducing the formation of human telomeric DNA G‐quadruplex structures, have not been reported. Herein, the interaction between single‐strand human telomeric DNA and three QBAs: Sanguinarine (San), Nitidine (Nit) and Chelerythrine (Che), has been investigated. Although these molecules are very similar in structure, they exhibit significantly different abilities in inducing oligonucleotide d(TTAGGG)₄ (HT4) to specific G‐quadruplex structures. Our experimental results indicated that the best ligand San could convert HT4 into antiparallel G‐quadruplex structure completely, followed by Nit, which could transform to mixed‐type or hybrid G‐quadruplex structure partially, whereas Che could only transform to antiparallel G‐quadruplex structure in small quantities. The relative QBAs' inducing abilities as indicated by the CD data are in the order of San>Nit>Che. Further investigation revealed that the G‐quadruplex structures from HT4 induced by QBAs are of intramolecular motif. And only sequences with certain length could be induced by QBAs because of their positive charges which could not attract short chain DNA molecules to close to each other and form intermolecular G‐quadruplex. In addition, the factors that affect the interaction between HT4 and QBAs were discussed. It is proposed that the thickness of the molecular frame and the steric hindrance are the primary reasons why the subtle differences in QBAs' structure lead to their remarkable differences in inducing the formation of the G‐quadruplex structures.