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.     

Competition of Ligands and the 18-mer Binding Domain of the RHAU Helicase for G-Quadruplexes: Orthosteric or Allosteric Binding Mechanism?

Stabilizing the DNA and RNA structures known as G-quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G-quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G-quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G-quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu–ttpy (ttpy=4′-tolyl-2,2′:6′,2′′-terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug–target interactions, but also how the binding to other partners is affected.     

Identifying G-quadruplex-binding ligands using DNA-functionalized gold nanoparticles

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.     

Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents

Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.     

Stiff-Stilbene Ligands Target G-Quadruplex DNA and Exhibit Selective Anticancer and Antiparasitic Activity

G-quadruplex nucleic acid structures have long been studied as anticancer targets whilst their potential in antiparasitic therapy has only recently been recognized and barely explored. Herein, we report the synthesis, biophysical characterization, and in vitro screening of a series of stiff-stilbene G4 binding ligands featuring different electronics, side-chain chemistries, and molecular geometries. The ligands display selectivity for G4 DNA over duplex DNA and exhibit nanomolar toxicity against Trypasanoma brucei and HeLa cancer cells whilst remaining up to two orders of magnitude less toxic to non-tumoral mammalian cell line MRC-5. Our study demonstrates that stiff-stilbenes show exciting potential as the basis of selective anticancer and antiparasitic therapies. To achieve the most efficient G4 recognition the scaffold must possess the optimal electronics, substitution pattern and correct molecular configuration.     

Methods for investigating G-quadruplex DNA/ligand interactions

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.     

高选择性端粒G-四链体稳定性配体:9-O-多胺取代小檗碱衍生物的合成及活性评价

设计合成了3个多胺取代的小檗碱衍生物5a~5c, 并利用圆二色(CD)光谱、 荧光共振能量转移(FRET)熔点实验、 荧光光谱和聚合酶链反应(PCR)终止实验等手段研究了小檗碱衍生物5a~5c与端粒DNA的相互作用. 结果表明, 小檗碱衍生物5a~5c可以诱导端粒DNA序列形成反平行结构G-四链体, 显著地提高了端粒G-四链体的稳定性, 有效地抑制了端粒的扩增; 而与双链DNA的相互作用则很小, 是高选择性的端粒G-四链体配体.     

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity**

A facile imide coupling strategy for the one-step preparation of G-quadruplex ligands with varied core chemistries is described. The G-quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV-Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G-quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3 , which was based on a perylene scaffold and exhibited 40-fold selectivity for a telomeric G-quadruplex over duplex DNA. Surprisingly, a tetra-substituted flexible core, compound 11 , also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78-fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.     

Contribution of telomere G-quadruplex stabilization to the inhibition of telomerase-mediated telomere extension by chemical ligands

Inhibition of telomerase activity through stabilizing telomere G-quadruplex with small chemical ligands is emerging as a novel strategy for cancer therapy. For the large number of ligands that have been reported to inhibit telomerase activity, it is difficult to validate the contribution of G-quadruplex stabilization to the overall inhibition. Using a modified telomere repeat amplification protocol (TRAP) method to differentiate the telomere G-quadruplex independent effect from dependent ones, we analyzed several ligands that have high affinity and/or selectivity to telomere G-quadruplex. Our results show that these ligands effectively inhibited telomerase activity in the absence of telomere G-quadruplex. The expected G-quadruplex-dependent inhibition was only obvious for the cationic ligands at low K(+) concentration, but it dramatically decreased at physiological concentration of K(+). These observations demonstrate that the ligands are much more than G-quadruplex stabilizers with a strong G-quadruplex-irrelevant off-target effect. They inhibit telomerase via multiple pathways in which stabilization of telomere G-quadruplex may only make a minor or neglectable contribution under physiologically relevant conditions depending on the stability of telomere G-quadruplex under ligand-free conditions.     

Platinum phenanthroimidazole complexes as G-quadruplex DNA selective binders

Complexes that bind and stabilize G-quadruplex DNA structures are of significant interest due to their potential to inhibit telomerase and halt tumor cell proliferation. We here report the synthesis of the first Pt(II) G-quadruplex selective molecules, containing pi-extended phenanthroimidazole ligands. Binding studies of these complexes with duplex and quadruplex d(T(4)G(4)T(4))(4) DNA were performed. Intercalation to duplex DNA was established through UV/Vis titration, CD spectroscopy, and thermal denaturation studies. Significantly stronger binding affinity of these phenanthroimidazole Pt(II) complexes to G-quadruplex DNA was observed by UV/Vis spectroscopy and competitive equilibrium dialysis studies. Observed binding constants to quadruplex DNA were nearly two orders of magnitude greater than for duplex DNA. Circular dichroism studies show that an increase in pi-surface leads to a significant increase in the thermal stability of the Pt(II)/quadruplex DNA complex. The match in the pi-surface of these phenanthroimidazole Pt(II) complexes with quadruplex DNA was further substantiated by molecular modeling studies. Numerous favorable pi-stacking interactions with the large aromatic surface of the intermolecular G-quadruplex, and unforeseen hydrogen bonds between the ancillary ethylenediamine ligands and the quadruplex phosphate backbone are predicted. Thus, both biological and computational studies suggest that coupling the square-planar geometry of Pt(II) with pi-extended ligands results in a simple and modular method to create effective G-quadruplex selective binders, which can be readily optimized for use in telomerase-based antitumor therapy.     

Binding of malachite green promotes stability and shows preference for a human telomere DNA G-quadruplex

A single-stranded human telomere DNA sequence can fold into an intramolecular G-quadruplex structure, which has been shown to inhibit telomerase activity. Small molecules that selectively target and stabilise the G-quadruplex structure have been proposed as potential anticancer drugs. In this study, we analysed the properties of binding of malachite green, a cationic triphenylmethane dye, to the G-quadruplex of d[(T₂AG₃)₄] by UV spectroscopy of thermal melting analysis, a competitive equilibrium dialysis assay, and absorption and circular dichroism spectroscopies. When binding to malachite green, the quadruplex structure that formed in the presence of K⁺ ions was stabilised with an increase in melting temperatures by 6 °C. Malachite green showed selective binding to the G-quadruplex in the presence of duplex and single-stranded DNAs, owing to which it presents higher potential for anticancer therapy, compared to other triphenylmethane dyes. The induced signals of circular dichroism indicate that the binding mode of malachite green involves intercalation between adjacent guanine tetrads of the G-quadruplex.     

咪唑修饰萘酰亚胺与DNA的作用及其细胞毒性

设计合成了咪唑及其烷基化咪唑阳离子基团修饰的萘酰亚胺衍生物。利用紫外-可见吸收光谱、荧光光谱、圆二色谱和荧光共振能量转移等方法研究了它们与小牛胸腺DNA(CT DNA)和G-四链体DNA的相互作用。这些化合物对端粒DNA序列的G-四链体有很高的结合能力(K_α4×10~6 L·mol~(-1)),并能够稳定G-四链体。DNA粘度实验结果表明萘酰亚胺衍生物与CT DNA通过插入作用结合。Autodock分子对接模拟结果表明这些化合物通过疏水作用、静电作用或氢键等方式与人体端粒G-四链体的loop和沟槽部分结合。咪唑阳离子基团修饰的萘酰亚胺衍生物4a–c能够定位于细胞核,对肺癌细胞的细胞毒性要高于咪唑基团修饰的萘酰亚胺衍生物3。化合物4a和4b对肺癌细胞A549的细胞毒性明显高于正常人胚肺成纤维细胞MRC-5,表现出良好的抗癌活性。     

A platinum supramolecular square as an effective G-quadruplex binder and telomerase inhibitor

In this contribution, we report that a self-assembled platinum molecular square [Pt(en)(4,4'-dipyridyl)]4 can act as an efficient G-quadruplex binder and telomerase inhibitor. Molecular modeling studies show that the square arrangement of the four bipyridyl ligands, the highly electropositive nature of the overall complex, as well as hydrogen bonding interactions between the ethylenediamine ligands and phosphates of the DNA backbone all contribute to the observed strong binding affinity to the G-quadruplex. Through thermal denaturation studies with duplex and quadruplex FRET probes and enzymatic assays, we demonstrate that this platinum square strongly binds to G-quadruplexes and can act as an inhibitor of telomerase. This study thus shows the potential of supramolecular self-assembly to readily generate scaffolds of unique geometries for effective targeting of G-quadruplexes and for the ultimate development of selective antitumor therapies.     

G4 Sensing Pyridyl-Thiazole Polyamide Represses c-KIT Expression in Leukemia Cells

Specific sensing and functional tuning of nucleic acid secondary structures remain less explored to date. Herein, we report a thiazole polyamide TPW that binds specifically to c-KIT1 G-quadruplex (G4) with sub-micromolar affinity and ∼1 : 1 stoichiometry and represses c-KIT proto-oncogene expression. TPW shows up to 10-fold increase in fluorescence upon binding with c-KIT1 G4, but shows weak or no quantifiable binding to other G4s and ds26 DNA. TPW can increase the number of G4-specific antibody (BG4) foci and mark G4 structures in cancer cells. Cell-based assays reveal that TPW can efficiently repress c-KIT expression in leukemia cells via a G4-dependent process. Thus, the polyamide can serve as a promising probe for G-quadruplex recognition with the ability to specifically alter c-KIT oncogene expression.     

Macrocyclization of bis-indole quinolines for selective stabilization of G-quadruplex DNA structures

The recognition of G-quadruplex (G4) DNA structures as important regulatory elements in biological mechanisms, and the connection between G4s and the evolvement of different diseases, has sparked interest in developing small organic molecules targeting G4s. However, such compounds often lack drug-like properties and selectivity. Here, we describe the design and synthesis of a novel class of macrocyclic bis-indole quinolines based on their non-macrocyclic lead compounds. The effects of the macrocyclization on the ability to interact with G4 DNA structures were investigated using biophysical assays and molecular dynamic simulations. Overall, this revealed compounds with potent abilities to interact with and stabilize G4 structures and a clear selectivity for both G4 DNA over dsDNA and for parallel/hybrid G4 topologies, which could be attributed to the macrocyclic structure. Moreover, we obtained knowledge about the structure–activity relationship of importance for the macrocyclic design and how structural modifications could be made to construct improved macrocyclic compounds. Thus, the macrocyclization of G4 ligands can serve as a basis for the optimization of research tools to study G4 biology and potential therapeutics targeting G4-related diseases.

Macrocyclization improves the selectivity, affinity, and ability to stabilize G4 DNA structures.     

Platinum(II) complexes with dipyridophenazine ligands as human telomerase inhibitors and luminescent probes for G-quadruplex DNA

A series of platinum(II) complexes containing dipyridophenazine (dppz) and C-deprotonated 2-phenylpyridine (N-CH) ligands were prepared and assayed for G-quadruplex DNA binding activities. [PtII(dppz-COOH)(N-C)]CF3SO3 (1; dppz-COOH = 11-carboxydipyrido[3,2-a:2',3'-c]phenazine) binds G-quadruplex DNA through an external end-stacking mode with a binding affinity of approximately 10(7) dm3 mol-1. G-quadruplex DNA binding is accompanied by up to a 293-fold increase in the intensity of photoluminescence at lambdamax = 512 nm. Using a biotinylated-primer extension telomerase assay, 1 was shown to be an effective inhibitor of human telomerase in vitro, with a telIC50 value of 760 nM.     

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。     

Stabilization and structure of telomeric and c-myc region intramolecular G-quadruplexes: the role of central cations and small planar ligands

A promising approach for anticancer strategies is the stabilization of telomeric DNA into a G-quadruplex structure. To explore the intrinsic stabilization of folded G-quadruplexes, we combined electrospray ionization mass spectrometry, ion mobility spectrometry, and molecular modeling studies to study different DNA sequences known to form quadruplexes. Two telomeric DNA sequences of different lengths and two DNA sequences derived from the NHE III1 region of the c-myc oncogene (Pu22 and Pu27) were studied. NH4+ and the ligands PIPER, TMPyP4, and the three quinacridines MMQ1, MMQ3, and BOQ1 were complexed with the DNA sequences to determine their effect on the stability of the G-quadruplexes. Our results demonstrate that G-quadruplex intramolecular folds are stabilized by NH4+ cations and the ligands listed. Furthermore, the ligands can be classified according to their ability to stabilize the quadruplexes and end stacking is shown to be the dominant mode for ligand attachment. In all cases our solvent-free experimental observations and theoretical modeling reveal structures that are highly relevant to the solution-phase structures.     

Electrochemical investigation on the interaction of benzene sulfonyl 5-fluorouracil derivatives with double-stranded DNA and G-quadruplex DNA

The interaction of double-stranded(ds) and G-quadruplex(G4) DNA with sulfonyl 5-fluorouracil derivatives(5-fluoro-1-(arylsulfonyl) pyrimidine-2,4(1H,3H)-diones) was investigated in this research,in which Au electrodes modified with ds-DNA or G4-DNAs were used as a working electrode.The investigation showed that the binding affinity with G4-DNA was significantly increased when 5-fluorouracil(5-FU) was modified with arylsulfonyl groups.The presence of strong electron-withdrawing groups on benzene sulfonyl 5-FU greatly enhanced the binding selectivity(k G4-DNA /k ds-DNA).Such results provided new insights into the potential connections between the chemical structure of drug candidates and their anticancer activities.     

Impact of planarity of unfused aromatic molecules on G-quadruplex binding: learning from isaindigotone derivatives

G-quadruplex structures are a new class of attractive targets for DNA-interactive anticancer agents. The primary building block of this structure is the G-quartet, which is composed of four coplanar guanines and serves as the major binding site for small molecules. NMR studies and molecular dynamics simulations have suggested that the planarity of G-quartet surface has been highly dynamic in solution. To better investigate how the planarity of unfused aromatic ligand impacts on its quadruplex binding properties, a variety of planarity controllable isaindigotone derivatives were designed and synthesized. The interaction of G-quadruplex DNA with these designed ligands was systematically explored using a series of biophysical studies. The FRET-melting, SPR, and CD spectroscopy results showed that reducing the planarity of their unfused aromatic core resulted in their decreased binding affinity and stabilization ability for G-quadruplex. NMR studies also suggested that these compounds could stack on the G-quartet surface. Such results are in parallel with subsequent molecular modeling studies. A detailed binding energy analysis indicated that van der Waals energy (ΔE(vdw)) and entropy (TΔS) are responsible for their decreased quadruplex binding and stabilization effect. All these results provided insight information about how quadruplex recognition could be controlled by adjusting the planarity of ligands, which shed light on further development of unfused aromatic molecules as optimal G-quadruplex binding ligands.