G四链体晶体结构及其小分子配体的研究进展

端粒酶能阻止端粒的缩短使细胞无限增殖形成肿瘤，G四链体是端粒末端单链在一定生理条件下形成的具有抑制端粒酶活性的结构，能稳定G四链体结构的配体具有重要的抗肿瘤意义。熟悉和了解G四链体的晶体结构，以及与配体的作用模式，有助于合理设计抗肿瘤药物。本文综述了目前已知的G四链体结构及其与配体的作用模式，以及小分子配体的研究进展。     

人体端粒G4-DNA稳定剂的研究进展

端粒长度的维持在肿瘤细胞的永生化过程中起到至关重要的作用。约85%的人体肿瘤细胞通过端粒酶延伸端粒,从而获得持续的增殖能力。另外,15%的人体肿瘤细胞通过端粒替代延伸机制(alternative lengthening of telomeres,ALT)延伸端粒。这两种机制对于维持肿瘤细胞中端粒的长度具有同等重要的意义。人体端粒由富含鸟嘌呤(G)的DNA重复序列组成,该序列在特定的条件下可以形成G-四链体(G4)的结构。此结构的形成可以从根本上抑制端粒酶和ALT对端粒的延伸而达到抗肿瘤的目的。因此,人体端粒G4-DNA作为抗肿瘤靶点的研究是近年来抗肿瘤研究的重要前沿领域之一。该文重点综述人体端粒G4-DNA稳定剂研发的最新研究进展。     

金属配合物与G-四链体作用的研究进展

端粒DNA形成的独特二级结构G-四链体对端粒酶的活性有明显抑制作用,能达到促进肿瘤细胞凋亡的作用。研究表明,能够诱使端粒DNA形成G-四链体及可以稳定这种结构的药物均可能成为有效的化疗药物。金属配合物由于其合成路径简单、结构多变、金属中心所携带的正电荷有利于与G-四链体的沟区、loop环以及带负电荷的磷酸骨架作用,增强其稳定性,因此越来越多的过渡金属配合物被设计用来研究与G-四链体作用。该文综述了近年来金属配合物与G-四链体DNA相互作用方面的研究进展。     

MG与人类端粒DNA G-四链体相互作用的研究

目的:研究MG对人类端粒DNA G-四链体的稳定作用,为发展新型抗癌药物提供理论依据。方法:采用紫外光谱法和荧光光谱法研究MG与端粒DNA G-四链体的相互作用。结果:MG与杂化型端粒DNA G-四连体有很好的亲和性。结论:MG作为平面共轭的小分子配体与端粒DNA G-四链体良好的结合能力可以为修饰和设计更加优良的端粒DNA G-四链体配体提供依据。     

基于分子对接研究黄酮及异黄酮类化合物与G-四链体DNA的相互作用

摘要：目的:探讨端粒G-四链体DNA稳定剂黄酮及异黄酮类化合物的作用模式,进而确立比较可靠的化合物分子对接模型用于后续研究。方法:以两个具有与黄酮及异黄酮相似结构配体的G-四链体DNA晶体为受体,将文献中报道了活性的黄酮及异黄酮类化合物经过能量优化后作为配体,采用SYBYL-X 2.0的对接模块进行分子对接。通过对接总评分与活性数据对比来分析对接模型的合理性,同时研究化合物与G-四链体DNA的相互作用。结果:分子对接软件计算出的总评分与化合物活性数据比较匹配,预测的受体-配体间相互作用对化合物结构优化具有较好的参考意义。结论:通过分子模拟及结果分析建立了比较合理的对接模型,在下一步工作中可应用该模型进行虚拟筛选,指导化合物设计。     

以KRAS启动子G-四链体DNA为靶点的血根碱对人结肠癌细胞SW620作用及其机制

目的　研究血根碱与KRAS 启动子G- 四链体DNA 的相互作用,探究以KRAS G- 四链体DNA 为靶点的血根碱对人结肠癌细胞SW620 的作用及机制。方法　采用MTT 法检测血根碱对人结肠癌细胞SW620 增殖的影响。采用紫外- 可见吸收光谱、荧光光谱、圆二色谱等方法研究血根碱与KRAS 启动子G- 四链体DNA 的相互作用。采用实时荧光定量PCR（Real-Time PCR）检测血根碱对SW620 细胞KRAS mRNA 表达的影响。结果　血根碱能够抑制人结肠癌SW620 细胞的增殖。血根碱与KRAS 启动子G- 四链体DNA 的结合能力较强,结合计量比接近1∶1,结合分子机制为生成了不发光的复合物。血根碱可维持G- 四链体DNA 的平行构型及热稳定性。血根碱能抑制人结肠癌SW620 细胞KRAS mRNA 的表达。结论　血根碱可能通过与KRAS 启动子区域G- 四链体结合并维持其结构的稳定,抑制KRAS mRNA 表达,进而抑制人结肠癌细胞SW620 的增殖,发挥抗癌作用。     

G—四链体DNA：抗肿瘤药物的潜在靶点

除了常见的双链DNA,某些富含G碱基的DNA序列通过四个鸟嘌呤环的互联作用可折叠成四链螺旋结构,这样的DNA二级结构被称为G－四链体。本文综述了G－四链体可能在生物学么及作为抗肿瘤药物潜在靶点能与其相互作用的化合物的研究与开发。     

荷包牡丹碱对人肺癌A549细胞生长及端粒酶活性的抑制作用

目的探讨荷包牡丹碱对人肺癌A549细胞生长的抑制作用及其作用机制。方法 A549细胞加入荷包牡丹碱0～200μmol.L-1分别作用24,48和72 h,MTT法测定A549细胞的生长抑制作用。荷包牡丹碱0～20μmol.L-1作用72 h,端粒重复序列扩增(TRAP)法测定端粒酶活性。变温紫外法检测荷包牡丹碱9μmol.L-1对端粒酶G-四链体的稳定作用。结果荷包牡丹碱25,50,100和200μmol.L-1作用细胞72 h后的抑制率分别为33.4%,88.2%,88.6%和89.4%,明显高于正常对照组细胞(P<0.05),并具有量效(r=0.906,P<0.05)和时效(r=0.949,P<0.05)性。与正常对照组相比,荷包牡丹碱5,10,15和25μmol.L-1可有效抑制A549细胞端粒酶的活性(P<0.05),相对TRAP端粒酶活性从正常对照组的1.471±0.102分别降低为1.093±0.054,1.013±0.016,0.554±0.034,0.365±0.081(P<0.05)。荷包牡丹碱9μmol.L-1使G-四链体的熔点值从正常对照组的48℃提高到54℃。结论荷包牡丹碱可以通过稳定G-四链体结构,抑制端粒酶活性,有效抑制人肺腺癌细胞A549的生长。     

G-四链体的结构及其与小分子配体成复合物的结构研究进展

通过与DNA的结合而从生物学源头阻止疾病的发生成为近年研究的热点,DNA G-四链体结构的发现以及分子生物学相关技术对其与癌症关系的揭示,为抗肿瘤药物的设计提供了一个良好的突破口。本文综述了近三年来G-四链体的结构及其与小分子配体成复合物的结构研究进展,有助于理解G-四链体与小分子配体的作用模式,以期为合理设计抗肿瘤药物有所帮助。     

浙贝碱与DNA G-四链体复合物的分子动力学模拟研究

目的:探讨浙贝碱与平行型DNA G-四链体的结合特征,为发展新型DNA G-四链体稳定剂提供理论基础.方法:采用分子对接方法确定浙贝碱在平行型DNA G-四链体中的初始结合位置,通过动力学模拟寻找平衡结构.结果:浙贝碱能够选择性的作用于平行型DNA G-四链体的沟槽与loop共同形成的位点,并在动力学过程中稳定存在.结...     

G-Quadruplex DNA as a target for drug design

Telomeres are structures on the ends of chromosomes that are required for chromosomal stability. Telomeric DNA contains a single-stranded G-rich DNA overhang, which may adopt a G-quadruplex structure. Telomere shortening has been implicated in cellular senescence. Telomerase is an enzyme which synthesizes the G-rich strand of telomere DNA. Telomerase activity is highly correlated with cancer and may allow cancer cells to escape senescence. Based on these observations, telomerase has been proposed as a potential target for anticancer drug design. The targeting of telomerase is associated with potential problems, including the existence in some cancer cells of telomerase-independent mechanisms for telomere maintenance, and the long delay time between telomerase inhibition and effects on proliferation. One promising approach for inhibiting telomerase involves targeting the G-quadruplex DNA structures thought to be involved in telomere and telomerase function. Compounds that specifically bind G-quadruplex DNA may interact directly with telomeres, in addition to inhibiting telomerase, and produce more immediate antiproliferative effects. The diamidoanthraquinones, porphyrins, and perylene diimides have all been shown to bind G-quadruplex DNA and inhibit telomerase. Most of these compounds also bind double-stranded DNA and are cytotoxic at the concentrations required to inhibit telomerase; however, certain perylene diimides appear to be non-cytotoxic, G-quadruplex selective telomerase inhibitors. Biological characterization of such compounds may provide validation for the concept of the G-quadruplex as a target in drug design.     

Tetraplex binding molecules as anti-cancer agents

Increasing evidence suggests that four-stranded tetraplex structures spontaneously form under physiological conditions and these alternate DNA structures are likely to form in vivo. Alternate G-quadruplex DNA structures that may form in regulatory elements of oncogenes or G-rich telomere sequences are potential targets for cancer therapy since these sequence-specific structures are proposed to affect gene expression and telomerase activation, respectively. Small molecule compounds that specifically bind tetraplexes may be used to regulate cell cycle progression by modulating promotor activation or disrupting telomere maintenance, important processes of cellular transformation. In this review, we summarize the current research developments and associated patents that bear relevance to understanding the mechanism and clinical application of tetraplex binding molecules as anti-cancer agents.     

Synthesis and evaluation of quindoline derivatives as G-quadruplex inducing and stabilizing ligands and potential inhibitors of telomerase

A new series of quindoline derivatives (4a-j) were designed and synthesized to develop novel and potent telomerase inhibitors. The interaction of the G-quadruplex of human telomere DNA with these newly designed molecules was examined via circular dichroism spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative (4a) and G-quadruplex or duplex DNA was investigated by competition dialysis. These new compounds as inhibitors of telomerase were also investigated through the utilization of modified telomerase repeat amplification protocol (TRAP) assay. The results revealed that the introduction of electron-donating groups such as substituted amino groups at the C-11 position of quindoline significantly improved the inhibitory effect on telomerase activity ((Tel)IC50 > 138 microM for quindoline, 0.44-12.3 microM for quindoline derivatives 4a-j). The quindoline derivatives not only stabilized the G-quadruplex structure but also induced the G-rich telomeric repeated DNA sequence to fold into quadruplex.     

Telomeres and telomerase, new targets for anticancer chemotherapy

Telomeres are composed of single-strand DNA rich in guanine which can adopt particular structures such as T-loop or G-quadruples, a four-strand DAN structure formed by guanine repeats. Telomeric single-strand DNA is the substrate of telomerase, an enzyme necessary for telomeric replication which is suppressed in most cancer cells and which participates in tumor genesis. The formation of a telomeric G-quadruplex blocks telomerase activity and offers an original strategy for new anti-cancer agents. Using an original approach combining rational screening and synthesis, several series of compounds have been identified which specifically bind to the telomeric quadruplex. These derivatives, called "G-quadruplex DNA ligands", are able to block telomeric replication in cancer cells and provoke replicative senescence and/or apoptosis after a few cell cycles. Our team is working on characterizing the cellular and molecular mechanisms of action of these ligands. Using mutant cell models resistant to these ligands or expressing a protein cuff covering the telomere in tumor lines, we have demonstrated that the telomere is the principal intracellular target of action of these compounds and the implicit existence of the G-quadruplex structure. In collaboration with academic and industrial partners, optimization of these ligands to develop pharmacologically active products should enable in vivo validation of a new therapeutic concept.     

Remarkable interference with telomeric function by a G-quadruplex selective bisantrene regioisomer

The use of small molecules able to induce and stabilize selected G-quadruplex arrangements can cause telomerase inhibition and telomere dysfunction in cancer cells, thus providing very selective therapeutic approaches. Effective stabilizers usually comprise a planar aromatic portion to grant effective stacking onto the G-quartet and positively charged side chains to exploit the highly negative charge density on the quadruplex grooves. Since the relative position of these two pharmacophoric moieties is expected to play an important role in DNA folding stabilization, we evaluated a series of anthracene derivatives substituted with one or two 4,5-dihydro-1H-imidazol-2-yl-hydrazonic groups (the bisantrene side chain) at different positions of the aromatic system. Indeed, the various regioisomers showed distinct binding affinities for telomeric G-quadruplex, and the most effective was the 1,5 and 1,7 bis-substituted analogues. On turn, the 1,8 regioisomer was poorly effective. Interestingly, G-quadruplex binding is clearly related to telomerase inhibition in this class of compounds, thus confirming their ability to shift the nucleic acid conformational equilibrium upon binding and consequently produce interference with the telomere processing enzyme. Additionally, the 1,5 regioisomer was shown to inhibit telomerase activity at lower concentrations than those required to reduce tumor cell proliferation. Comparative analysis of drug effects in telomerase-positive and telomerase-negative cancer cells showed consistent cell growth impairment, as a consequence of activation of the senescence pathway, which was mainly attributable to anthracene-mediated telomere dysfunction.     

Searching drug-like anti-cancer compound(s) based on G-quadruplex ligands

G-quadruplex structure is a four-stranded form of DNA, which is associated with cancer cell proliferation. G-quadruplex-stabilized ligands have the potential to interfere with telomere replication by blocking the elongation procedure catalyzed by telomerase, and therefore have the potential to be anti-cancer drugs. A considerable number of novel compounds capable of targeting G-quadruplex at high affinity and specificity have been reported. Among them, several G-quadruplex ligands have shown promising anti-cancer activity in tumor xenograft models, and entered phase II clinical trials on cancer patients. This review summarized recent developments of G-quadruplex ligands as anti-cancer drugs and several powerful strategies to discover novel G-quadruplex ligands as anti-cancer drug candidates by screening natural product extracts and structural optimization of previously identified typical compounds.     

G-quadruplex interacting agents targeting the telomeric G-overhang are more than simple telomerase inhibitors

The extremities of chromosomes end at telomeres in a G-rich single stranded overhang that may adopt peculiar structures such as T-loop and G-quadruplex. G-quadruplex is a poor substrate for telomerase activity and different classes of small molecule ligands that selectively stabilize this structure and inhibit telomerase activity have been selected by screening or synthesized by oriented chemistry. These ligands differ from catalytic inhibitors of telomerase by several points that were discussed in the present review, with a special emphasis on their biological activity as potential antitumor agents.     

A new steroid derivative stabilizes g-quadruplexes and induces telomere uncapping in human tumor cells

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) with a 3' single-stranded extension (the G-overhang). The stabilization of G-quadruplexes in the human telomeric sequence by small-molecule ligands inhibits the activity of telomerase and results in telomere uncapping, leading to senescence or apoptosis of tumor cells. Therefore, the search for new and selective G-quadruplex ligands is of considerable interest because a selective ligand might provide a telomere-targeted therapeutic approach to treatment of cancer. We have screened a bank of derivatives from natural and synthetic origin using a temperature fluorescence assay and have identified two related compounds that induce G-quadruplex stabilization: malouetine and steroid FG. These steroid derivatives have nonplanar and nonaromatic structures, different from currently known G-quadruplex ligands. Malouetine is a natural product isolated from the leaves of Malouetia bequaaertiana E. Woodson and is known for its curarizing and DNA-binding properties. Steroid FG, a funtumine derivative substituted with a guanylhydrazone moiety, interacted selectively with the telomeric G-quadruplex in vitro. This derivative induced senescence and telomere shortening of HT1080 tumor cells at submicromolar concentrations, corresponding to the phenotypic inactivation of telomerase activity. In addition, steroid FG induced a rapid degradation of the telomeric G-overhang and the formation of anaphase bridges, characteristics of telomere uncapping. Finally, the expression of protection of telomere 1 (POT1) induced resistance to the growth effect of steroid FG. These results indicate that these steroid ligands represent a new class of telomere-targeted agents with potential as antitumor drugs.     

Energetics of quadruplex-drug recognition in anticancer therapy

Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpressed in about 85% of tumour cells and maintains telomere length contributing to cell immortalisation, whereas in somatic cells telomeres progressively shorten until cell death occurs by apoptosis. Different drugs can promote telomeric G-rich overhangs which fold into quadruplex structures that inhibit telomerase activity. Detailed studies on drug-quadruplex complexes are essential to understand quadruplex recognition and address drug design. This review will discuss the energetic aspects of quadruplex-drug interactions with a particular attention to physico-chemical methodologies.