端粒G-四联体及其在肿瘤治疗中的应用

G-四联体是端粒形成的一种特殊的结构.如果药物能稳定G-四联体结构或促使其形成,则可使端粒酶不能发挥其逆转录酶活性,不仅抑制端粒酶活性而且使端粒不能延伸.而恶性肿瘤需要一定的端粒长度才能维持其生长及增殖,所以通过促成G-四联体结构的药物可以达到抑制恶性肿瘤生长的目的.本文G-四联体结构及其在肿瘤治疗中的应用作一综述.     

Interaction of an acridine dimer with DNA quadruplex structures.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.     

Design,synthesis and evaluation of 4,5-di-substituted acridone ligands with high G-quadruplex affinity and selectivity,together with low toxicity to normal cells

A series of 4,5-di-substituted acridones have been designed and synthesized. Several compounds show high affinity for telomeric G-quadruplex DNA in classical and competition FRET assays, together with low duplex DNA affinity, although they do not show activity in a telomerase assay or evidence of telomere shortening. They have low toxicity against a panel of cancer cell lines and a normal human fibroblast line, and produce potent senescence-based long-term growth arrest in the MCF7 and A549 cancer cell lines.     

Nitric oxide releasing acridone carboxamide derivatives as reverters of doxorubicin resistance in MCF7/Dx cancer cells

A series of nitric oxide donating acridone derivatives are synthesized and evaluated for in vitro cytotoxic activity against different sensitive and resistant cancer cell lines MCF7/Wt, MCF7/Mr (BCRP overexpression) and MCF7/Dx (P-gp expression). The results showed that NO-donating acridones are potent against both the sensitive and resistant cells. Structure activity relationship indicate that the nitric oxide donating moiety connected through a butyl chain at N¹⁰ position as well as morpholino moiety linkage through an amide bridge on the acridone ring system at C-2 position, are required to exert a good cytotoxic effect. Further, good correlations were observed when cytotoxic properties were compared with in vitro nitric oxide release rate, nitric oxide donating group potentiated the cytotoxic effect of the acridone derivatives. Exogenous release of nitric oxide by NO donating acridones enhanced the accumulation of doxorubicin in MCF7/Dx cell lines when it was coadministered with doxorubicin, which inhibited the efflux process of doxorubicin. In summary, a nitric oxide donating group can potentiate the anti-MDR property of acridones.     

Structure-based design of benzylamino-acridine compounds as G-quadruplex DNA telomere targeting agents

The design, synthesis, biophysical and biochemical evaluation is presented of a new series of benzylamino-substituted acridines as G-quadruplex binding telomerase inhibitors. Replacement of the previously reported anilino substituents by benzylamino groups results in enhanced quadruplex interaction, and for one compound, superior telomerase inhibitory activity.     

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.     

Studies on the binding of 5-N-methylated quindoline derivative to human telomeric G-quadruplex

Quindoline derivatives as telomeric quadruplex ligands have shown good biological activity for telomerase inhibition. In the present study, we used spectroscopic and calorimetric methods to investigate the interactions between a quindoline derivative (5-methyl-11-(2-morpholinoethylamino)-10-H-indolo-[3,2-b]quinolin-5-ium iodide, compound 1) and human telomeric G-quadruplex. The thermodynamic studies using isothermal titration calorimetry (ITC) indicated that their binding process was temperature-dependent and enthalpy–entropy co-driven. The significant negative heat capacity was obtained experimentally from the temperature dependence of enthalpy changes, which was consistent with that from theoretical calculation, and all suggesting significant hydrophobic contribution to the molecular recognition process. Based on the results from UV–vis, ITC, steady-state and time-resolved fluorescence, their binding mode was determined as two ligand molecules stacking on the quartets on both ends of the quadruplex. These results shed light on rational design and development of quindoline derivatives as G-quadruplex binding ligands.     

Exploring the recognition of quadruplex DNA by an engineered Cys2-His2 zinc finger protein

We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.     

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.     

Drug–DNA Interaction Studies of Acridone-Based Derivatives

N¹⁰-alkylated 2-bromoacridones are a novel series of potent antitumor compounds. DNA binding studies of these compounds were carried out using spectrophotometric titrations, Circular dichroism (CD) measurements using Calf Thymus DNA (CT DNA). The binding constants were identified at a range of K = 0.3 to 3.9 × 10⁵ M^?1 and the percentage of hypochromism from the spectral titrations at 28–54%. This study has identified a compound 9 with the good binding affinity of K = 0.39768 × 10⁵ M^?1 with CT DNA. Molecular dynamics (MD) simulations have investigated the changes in structural and dynamic features of native DNA on binding to the active compound 9. All the synthesized compounds have increased the uptake of Vinblastine in MDR KBCh^R-8-5 cells to an extent of 1.25- to1.9-fold than standard modulator Verapamil of similar concentration. These findings allowed us to draw preliminary conclusions about the structural features of 2-bromoacridones and further chemical enhancement will improve the binding affinity of the acridone derivatives to CT-DNA for better drug–DNA interaction. The molecular modeling studies have shown mechanism of action and the binding modes of the acridones to DNA.     

Benzofuroquinoline Derivatives Had Remarkable Improvement of their Selectivity for Telomeric G-Quadruplex DNA over Duplex DNA upon Introduction of Peptidyl Group

In order to improve the selectivity of 5-N-methyl quindoline (cryptolepine) derivatives as telomeric quadruplex binding ligands versus duplex DNA, a series of peptidyl-benzofuroquinoline (P-BFQ) conjugates (2a-2n) were designed and synthesized. Their interactions with telomeric quadruplex and duplex DNA were examined by using the fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism spectroscopy (CD), and molecular modeling studies. Introduction of a peptidyl group at 11-position of the aromatic benzofuroquinoline scaffold not only effectively increased its binding affinity, but also significantly improved its selectivity toward telomeric quadruplex versus duplex DNA. Combined with the data for their inhibitory effects on telomerase activity, their structure-activity relationships (SARs) studies showed that the types of amino acid residues and the length of the peptidyl side chains were important for the improvement of their interactions with the telomeric G-quadruplex. Long-term exposure of human cancer cells to 2c showed a remarkable cessation in population growth and cellular senescence phenotype, and accompanied by a shortening of the telomere length.     

An exonuclease I hydrolysis assay for evaluating G-quadruplex stabilization by small molecules

Telomere length homeostasis is a prerequisite for the generation and growth of cancer. In >85% cancer cells, telomere length is maintained by telomerase that add telomere repeats to the end of telomere DNA. Because the G-rich strand of telomere DNA can fold into G-quadruplex that inhibits telomerase activity, stabilizing telomere quadruplex by small molecules is emerging as a potential therapeutic strategy against cancer. In these applications, the specificity of small molecules toward quadruplex over other forms of DNA is an important property to ensure no processes other than telomere elongation are interrupted. The evaluating assays currently available more or less have difficulty identifying or distinguishing quadruplex-irrelevant effect from quadruplex stabilization. Here, we describe an exonuclease I hydrolysis assay that evaluates quadruplex stabilization by DNA-interacting compounds, discriminates inhibitory effect from different sources and helps determine the optimal compound concentration.     

Synthesis of C8-linked pyrrolo[2,1-c][1,4]benzodiazepine-acridone/acridine hybrids as potential DNA-binding agents

Pyrrolobenzodiazepine hybrids linked to acridone/acridine ring systems at C8-position have been designed and prepared that exhibit significant DNA-binding affinity, and a representative compound shows promising in vitro anticancer activity.     

Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens

The root tips of Ruta graveolens (common rue) show strong autofluorescence of acridone alkaloids, which are characteristic secondary metabolites of this plant. To study the specific distribution and accumulation of acridone alkaloids in various root segments of Ruta graveolens, root material was harvested from genetically transformed root cultures and extracts were investigated by chromatographic techniques and HPLC-(1)H NMR spectroscopy. The cells of the elongation and differentiation zones contained acridone glucosides and large amounts of acridone alkaloids, mainly rutacridone. Gravacridondiol glucoside was identified as the dominant secondary compound of the root tips and its structure revised by means of spectroscopic methods. In addition, minor acridones, including the structurally revised gravacridontriol glucoside and unknown natural products, were found in the root tip.     

Acridone and furoquinoline alkaloids from Teclea gerrardii (Rutaceae: Toddalioideae) of southern Africa

The combined hexane/CH(2)Cl(2) extract of the stem bark of Teclea gerrardii (Rutaceae: Toddalioideae) has yielded two acridone alkaloids, 3-hydroxy-1-methoxy-N-methylacridone (tegerrardin A) (1) and 3-hydroxy-N-methyl-1-(gamma,gamma-dimethylallyloxy)acridone (tegerrardin B) (2), three known acridones (3-5), two known furoquinolines (6,7), and the acridone precursor tecleanone (8). Arborinine (3) and evoxine (6) displayed moderate antiplasmodial activity against the CQS D10 strain of Plasmodium falciparum, with IC(50) values of 12.3 and 24.5 microM, respectively.     

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI₅₀ 0.3 μM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI₅₀ in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.     

Aminoglycoside binding to Oxytricha nova telomeric DNA

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ～ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.     

Optimization of anti-proliferative activity using a screening approach with a series of bis-heterocyclic G-quadruplex ligands

Using a phenotypic screening and SAR optimization approach, a phenyl-bis-oxazole derivative has been identified with anti-proliferative activity, optimized with the use of a panel of cancer cell lines. The lead compound was synthesized by means of a short and effective two-step synthesis using Pd-catalyzed direct arylation. The compound stabilizes several quadruplex DNA sequences including a human telomeric DNA and one from the promoter of the HSP90 gene, although the structure–activity relationships of the series are not obviously related to the quadruplex binding.     

Synthesis and evaluation of acridine- and acridone-based anti-herpes agents with topoisomerase activity

The discovery of new non-nucleoside antiviral compounds is of significant and growing interest for treating herpes virus infections due to the emergence of nucleoside-resistant strains. Using a whole cell virus-induced cytopathogenic assay, we tested a series of substituted triaryl heterocyclic compounds including acridones, xanthones, and acridines. The compounds which showed activity against Herpes Simplex-1 and/or Herpes Simplex-2 were further assayed for inhibition of topoisomerase activity to gain insight into the mechanism of action. The results indicate that the acridine analogs bearing substituted carboxamides and bulky 9-amino functionalities are able to inhibit herpes infections as well as inhibit topoisomerase II relaxation of supercoiled DNA. Given the mechanism of action of amsacrine (a closely related, well-studied 9-amino substituted acridine), the compounds were further tested in a DNA topoisomerase II cleavage assay to determine if the compounds function as poisons. The results show that the acridines synthesized in this study function through a different mechanism to that of amsacrine, most likely by blocking topoisomerase binding to DNA (akin to that of aclarubicin). This not only suggests a unique mechanism of action in treating herpes virus infections, but also may be of great interest in the development of anticancer agents that target topoisomerase II activity.