Energetics of the human Tel-22 quadruplex-telomestatin interaction: a molecular dynamics study

The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadruplex as an attractive target for cancer therapeutic intervention. In this context, telomestatin, a G-quadruplex-specific ligand known to bind and stabilize G-quadruplex, is of great interest. Knowledge of the three-dimensional structure of telomeric quadruplex and its complex with telomestatin in solution is a prerequisite for structure-based rational drug design. Here, we report the relative stabilities of human telomeric quadruplex (AG3[T2AG3]3) structures under K+ ion conditions and their binding interaction with telomestatin, as determined by molecular dynamics simulations followed by energy calculations. The energetics study shows that, in the presence of K+ ions, mixed hybrid-type Tel-22 quadruplex conformations are more stable than other conformations. The binding free energy for quadruplex-telomestatin interactions suggests that 1:2 binding is favored over 1:1 binding. To further substantiate our results, we also calculated the change in solvent-accessible surface area (DeltaSASA) and heat capacity (DeltaCp) associated with 1:1 and 1:2 binding modes. The extensive investigation performed for quadruplex-telomestatin interaction will assist in understanding the parameters influencing the quadruplex-ligand interaction and will serve as a platform for rational drug design.     

Telomestatin: formal total synthesis and cation-mediated interaction of its seco-derivatives with G-quadruplexes

The structurally unique natural product telomestatin incorporates seven oxazole rings and one sulfur-containing thiazoline in a macrocyclic arrangement. The compound is a potent inhibitor of the enzyme telomerase and therefore provides a structural framework for developing new potential therapeutic agents for cancer. An efficient formal total synthesis of telomestatin is reported in which the key steps are the use of dirhodium(II)-catalyzed reactions of diazocarbonyl compounds to generate six oxazole rings, demonstrating the power of rhodium carbene methodology in organic chemical synthesis. CD spectroscopy establishes that seco-derivatives of telomestatin are potent stabilizers of G-quadruplex structures derived from the human telomeric repeat sequence. Mass spectrometry studies, confirmed by molecular dynamics simulations, provide the first evidence that high affinity binding to terminal G-tetrads in both 1:1 and 2:1 ligand complexes is mediated through the macrocycle coordinating a monovalent cation, with selectivity for the antiparallel structure.     

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.     

Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure

The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.     

(S)-stereoisomer of telomestatin as a potent G-quadruplex binder and telomerase inhibitor

Total synthesis of the (S)-stereoisomer of telomestatin (1) was accomplished. (S)-Telomestatin exhibited potency four times that of the natural product, (R)-telomestatin, which was the most potent telomerase inhibitor previously reported. In the circular dichroism spectral analysis of the complexes possessing randomly structured single-stranded d[TTAGGG](4) oligonucleotide, (S)-telomestatin, like (R)-telomestatin, induced an antiparallel G-quadruplex structure. The melting temperature (T(m)) value of the (S)-isomer complex was greater than that of the (R)-telomestatin complex. Therefore, it is concluded that the stereochemistry of the thiazoline of telomestatin is important to the binding ability of a G-quadruplex binder, and (S)-telomestatin as a G-quadruplex binder is more potent than the natural product.     

Disubstituted 2-phenyl-benzopyranopyrimidine derivatives as a new type of highly selective ligands for telomeric G-quadruplex DNA

A series of 2-phenyl-benzopyranopyrimidine (PBPP) derivatives with alkylamino side chains were synthesized and found to be a new type of highly selective ligand to bind with telomeric G-quadruplex DNA, and their biological properties were reported for the first time. Their interactions with telomeric G-quadruplex DNA were studied with FRET melting, surface plasmon resonance, CD spectroscopy, and molecular modeling. Our results showed that the disubstituted PBPP derivatives could strongly bind to and effectively stabilize the telomeric G-quadruplex structure, and had significant selectivity for G-quadruplex over duplex DNA. In comparison, the mono substituted derivatives had much less effect on the G-quadruplex, suggesting that the disubstitution of PBPP is essential for its interaction with the G-quadruplex. Furthermore, telomerase inhibition of the PBPP derivatives and their cellular effects were studied, and compound 11b was found to be the most promising compound as a telomerase inhibitor and telomeric G-quadruplex binding ligand for further development for cancer treatment.     

Pt(ii) squares as selective and effective human telomeric G-quadruplex binders and potential cancer therapeutics

A series of four self-assembled Pt(ii) molecular squares with 4,4'-dipyridyl or pyrazine bridges, including the previously reported Pt(ii) squares [Pt(en)(4,4'-dipyridyl)](4)(NO(3))(8) (1), were investigated for their abilities to act as selective and effective human telomeric (htelo) G-quadruplex binders. FRET and SPR studies demonstrated that Pt(ii) squares could discriminate against duplex DNA, and show promising selectivity between intramolecular G-quadruplexes. PCR-stop assays and CD studies showed that Pt(ii) squares strongly induced the formation of parallel G-quadruplexes. ITC experiments indicated that Pt(ii) squares could bind to the G-quadruplex with high binding constants (K(b) values ranging from 10(4)-10(8) M(-1)). All four Pt(ii) squares were effective inhibitors of human telomerase, and showed anticancer efficacy. This was particularly the case for [Pt(NH(3))(2)(4,4'-dipyridyl)](4)(NO(3))(8) (2), which exhibited a 15-fold higher antiproliferative effect on A549/cisR cells than cisplatin.     

Structure of the first parallel DNA quadruplex-drug complex

The first crystal structure of a drug (daunomycin) bound to a parallel-stranded intermolecular telomeric G4 quadruplex (d(TGGGGT)4) has been determined to high resolution. A planar assemblage of three daunomycin molecules stacks onto the 5' end of the G4 column, with the daunosamine substituents occupying three of the four quadruplex grooves. The surface area of the terminal G-quartet in this parallel DNA quadruplex, presently occupied by three daunomycins, is sufficiently large that it could easily accommodate other potential telomerase inhibitors such as substituted porphyrins or telomestatin.     

Neomycin-capped aromatic platforms: quadruplex DNA recognition and telomerase inhibition

A series of aminoglycoside-capped macrocyclic structures has been prepared using intramolecular bis-tethering of neomycin on three aromatic platforms (phenanthroline, acridine, quinacridine). Based on NMR and calculations studies, it was found that the cyclic compounds adopt a highly flexible structure without conformational restriction of the aminoglycoside moiety. FRET-melting stabilization measurements showed that the series displays moderate to high affinity for the G4-conformation of human telomeric repeats, this effect being correlated with the size of the aromatic moiety. In addition, a FRET competition assay evidenced the poor binding ability of all macrocycles for duplex DNA and a clear binding preference for loop-containing intramolecular G4 structures compared to tetramolecular parallel G4 DNA. Finally, TRAP experiments demonstrated that the best G4-binder (quinacridine ) is also a potent and selective telomerase inhibitor with an IC(50) in the submicromolar range (200 nM).     

Studies on characterization, telomerase inhibitory properties and G-quadruplex binding of η6-arene ruthenium complexes with 1,10-phenanthroline-derived ligands

Two arene ruthenium complexes [Ru(η(6)-C(6)H(6))(p-MOPIP)Cl](+)1 and [Ru(η(6)-C(6)H(6))(p-CFPIP)Cl](+)2, where p-MOPIP = 2-(4-methoxyphenyl)-imidazo[4,5f][1,10] phenanthroline and p-CFPIP = 2-(4-trifluoromethylphenyl)-imidazo[4,5f][1,10] phenanthroline, were prepared and the interactions of these compounds with DNA oligomers 5'-G3(T2AG3)3-3'(HTG21) have been studied by UV-vis and circular dichroism (CD) spectroscopy, gel mobility shift assay, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay and telomeric repeat amplification protocol (TRAP) assay. The results show that both complexes can induce the stabilization of quadruplex DNA but complex 1 is a better G-quadruplex binder than complex 2. The two ruthenium complexes tested led to an inhibition of the enzyme telomerase and complex 1 was the significantly better inhibitor. A novel visual method has been developed for making a distinction between G-quadruplex DNA and double DNA by our Ru complexes binding hemin to form the hemin-G-quadruplex DNAzyme. Furthermore, in vitro cytotoxicity studies showed complex 1 exhibited quite potent antitumor activities and the greatest inhibitory selectivity against cancer cell lines.     

Cyclo[n]pyrroles: size and site-specific binding to G-quadruplexes

Inhibiting the enzyme telomerase by stabilizing the G-quadruplex has potential in anticancer drug design. Diprotonated cyclo[n]pyrroles represent a set of expanded porphyrin analogues with structures similar to that of telomestatin, a natural product known to bind to and stabilize G-quadruplexes. As a first step toward testing whether cyclo[n]pyrroles display a similar function, a series of diprotonated cyclo[n]pyrroles (where n = 6, 7, and 8) was each added to the human telomere repeat sequence d(T(2)AG(3))(4) and examined with mass spectrometry, ion mobility, and molecular dynamics calculations. Nano-ESI-MS indicated that the smaller the cyclo[n]pyrrole, the more strongly it binds to the telomeric sequence. It was also found that cyclo[6]pyrrole bound to d(T(2)AG(3))(4) better than octaethylporphyrin, a finding rationalized by cyclo[6]pyrrole having a 2+ charge, while octaethylporphyrin bears no charge. Ion mobility measurements were used to measure the collision cross section of each d(T(2)AG(3))(4)/cyclo[n]pyrrole complex. Only one peak was observed in the arrival time distributions for all complexes, and the experimental cross sections indicated that only structures with d(T(2)AG(3))(4) in an antiparallel G-quadruplex arrangement and each cyclo[n]pyrrole externally stacked below the G-quartets occur under these experimental conditions. When the cyclo[n]pyrroles were intercalated or nonspecifically bound to the quadruplex, or if conformations different than antiparallel were considered for d(T(2)AG(3))(4), the theoretical cross sections did not match experiment. On this basis, it is inferred that (1) external stacking represents the dominant binding mode for the interaction of cyclo[n]pyrroles with d(T(2)AG(3))(4) and (2) the overall size and charge of the cyclo[n]pyrroles play important roles in defining the binding strength.     

Interaction between human telomere and a carbazole derivative: a molecular dynamics simulation of a quadruplex stabilizer and telomerase inhibitor

The mechanism of inhibition of telomerase by drugs is a key factor in an understanding of guanine-quadruplex complex stabilization during human cancer. This study describes a simulated annealing docking and molecular dynamics simulation to investigate a synthesized potent inhibitor, 3,6-bis(1-methyl-4-vinylpyridinium iodine) carbazole (BMVC), which stabilizes the quadruplex structure of the human telomeric DNA sequence d[AG3(T(2)AG(3))3] and inhibits telomerase activity. The compound was predicted to selectively interact with the quadruplex structure. During our simulation, the binding affinities were calculated and used to predict the best drug-binding sites as well as enhanced selectivity compared with other compounds. Our studies suggest that the simulation results quite coincide with the experimental results. In addition, molecular modeling shows that a 2:1 binding model involving the external binding of BMVC to both ends of the G-quartet of d[AG(3)(T(2)AG)3))3] is the most stable binding mode and this agrees with the absorbance titration results that show two binding sites. Of particular interest is that one pyridinium ring and carbazole moiety of the BMVC can stack well at the end of G-quartet. This implies that BMVC is a good human quadruplex stabilizer and also a good telomerase inhibitor.     

Telomestatin-induced stabilization of the human telomeric DNA quadruplex monitored by electrospray mass spectrometry

Electrospray mass spectrometry (ESI-MS) was used to monitor the kinetics of duplex formation between the human telomeric DNA quadruplex and its complementary strand; the complexation of telomestatin to the G-quadruplex delays the unwinding of the quadruplex structure and formation of the duplex.     

Characterization of structure and stability of long telomeric DNA G-quadruplexes

In the current study, we used a combination of gel electrophoresis, circular dichroism, and UV melting analysis to investigate the structure and stability of G-quadruplexes formed by long telomeric DNAs from Oxytricha and human, where the length of the repeat (n)=4 to 12. We found that the Oxytricha telomeric DNAs, which have the sequence (TTTTGGGG)n, folded into intramolecular and intermolecular G-quadruplexes depending on the ionic conditions, whereas human telomeric DNAs, which have the sequence (TTAGGG)n, formed only intramolecular G-quadruplexes in all the tested conditions. We further estimated the thermodynamic parameters of the intramolecular G-quadruplex. We found that thermodynamic stabilities of G-quadruplex structures of long telomeric DNAs (n=5 to 12) are mostly independent of sequence length, although telomeric DNAs are more stable when n=4 than when n>or=5. Most importantly, when n is a multiple of four, the change in enthalpy and entropy for G-quadruplex formation increased gradually, demonstrating that the individual G-quadruplex units are composed of four repeats and that the individual units do not interact. Therefore, we propose that the G-quadruplexes formed by long telomeric DNAs (n>or=8) are bead-on-a-string structures in which the G-quadruplex units are connected by one TTTT (Oxytricha) or TTA (human) linker. These results should be useful for understanding the structure and function of telomeres and for developing improved therapeutic agents targeting telomeric DNAs.     

A novel small molecule that alters shelterin integrity and triggers a DNA-damage response at telomeres

We describe a novel synthetic small molecule which shows an unprecedented stabilization of the human telomeric G-quadruplex with high selectivity relative to double-stranded DNA. We report that this compound can be used in vitro to inhibit telomerase activity and to uncap human POT1 (protection of telomeres 1) from the telomeric G-overhang. We also show that the small molecule G-quadruplex binder induces a partial alteration of shelterin through POT1 uncapping from telomeres in human HT1080 cancer cells and the presence of gammaH2AX foci colocalized at telomeres.     

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.     

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.     

Investigation of formation,recognition, stabilization,and conversion of dimeric G-quadruplexes of HIV-1 integrase inhibitors by electrospray ionization mass spectrometry

The dimeric G-quadruplex structures of d(GGGTGGGTGGGTGGGT) (S1) and d(GTGGTGGGTGGGTGGGT) (S2), the potent nanomolar HIV-1 integrase inhibitors, were detected by electrospray ionization mass spectrometry (ESI-MS) for the first time. The formation and conversion of the dimers were induced by NH(4)(+), DNA concentration, pH, and the binding molecules. We directly observed the specific binding of a perylene derivative (Tel03) and ImImImbetaDp in one system consisting of the intramolecular and the dimeric G-quadruplexes of the HIV-1 integrase inhibitor, which suggested that Tel03 could shift the equilibrium to the dimeric G-quadruplex formation, while ImImImbetaDp induces preferentially a structural change from the dimer to the intramolecular G-quadruplex. The results of this study indicated that Tel03 and ImImImbetaDp favor the stabilization of the dimeric G-quadruplex structures.     

Promoting the formation and stabilization of human telomeric G-quadruplex DNA, inhibition of telomerase and cytotoxicity by phenanthroline derivatives

Four new di-substituted phenanthroline-based compounds a-d have been designed and prepared, and they have been shown to induce the formation of anti-parallel structure of human telomeric G-quadruplex DNA by CD spectra. FRET assay indicates that the melting temperature increases (ΔT(m) values) of G-quadruplex in buffer (pH 7.4) containing 100 mM NaCl are 31.6, 34.6, 17.8 and 32.6 °C for the compounds (1.0 μM) a, b, c and d, respectively. Competitive FRET assay shows that the four compounds exhibit a high G-quadruplex DNA selectivity over duplex DNA. Three of the compounds are the potent telomerase inhibitors and HeLa cell proliferation inhibitors.