Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex

Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.     

Total synthesis of (R)-telomestatin

We have achieved a total synthesis of telomestatin, and its absolute configuration was determined to be (R). Coupling of cysteine-containing trisoxazole amine and serine-containing trisoxazole carboxylic acid, followed by macrocyclization, provided a 24-membered diamide. The seventh oxazole ring was formed by a Shin's procedure via dehydroamide. Cyclodehydration of a modified (R)-cysteine-(S-(t)Bu) moiety using Kelly's method (PPh3(O)-Tf2O) with anisole furnished (R)-telomestatin, whose CD spectrum was in good agreement with that of the natural product.     

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.     

A fragment based click chemistry approach towards hybrid G-quadruplex ligands: design,synthesis and biophysical evaluation

A library of hybrid oxazole–triazole based compounds containing contiguously linked aromatic units were synthesised as G-quadruplex binding ligands. The design of these ligands was based upon combining features of our first generation of G-quadruplex bis-triazole ligands and the natural product telomestatin. The syntheses and biophysical studies of these ligands are described.     

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-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.     

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.     

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.     

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.     

Convergent synthesis of a 24-membered macrocyclic hexaoxazole derivative related to the novel telomerase inhibitor telomestatin

An efficient construction of a 24-membered macrocyclic hexaoxazole derivative pertinent to the synthesis of analogues of the important natural product telomestatin was developed, which featured a convergent union of two trisoxazole units.     

电喷雾质谱法研究天然产物小分子识别人类端粒G-四链体及复合物的热稳定性

利用电喷雾质谱(ESI-MS)研究了12种天然产物小分子与人类端粒G-四链体结构的非共价相互作用和识别功能, 比较了不同小分子与人类端粒G-四链体的结合强弱, 发现了一种新的识别小分子——防己诺林碱对人类端粒G-四链体有很好的结合. 通过质谱升温实验比较了小分子结合对G-四链体热稳定性的影响, 防己诺林碱的结合使G-四链体的离子的解离温度(T1/2)上升到200 ℃. 利用分子模拟对G-四链体DNA与小分子结合的模式以及稳定性进行了探讨, 给出了防己诺林碱可能的结合位点和结合模式, Autodock计算出来的结合能约为-31.5 kJ·mol-1. 同原来的平面型分子不同, 防己诺林碱是一类新型结构的分子, 为设计合成新型G-四链体识别分子提供了新的结构模型.     

Direct screening of G-quadruplex ligands from Kalopanax septemlobus (Thunb.) Koidz extract by high performance liquid chromatography

G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, high-performance liquid chromatography (HPLC) was used for fast screening of G-quadruplex ligands from the crude extract of Kalopanax septemlobus (Thunb.) Koidz, a traditional Chinese medicine. Four potent G-quadruplex ligands were firstly selected through HPLC by comparing the peak profiles and absorption intensity of the crude sample before and after interaction with G-quadruplex DNA. Then the target compounds were isolated and purified by high-speed countercurrent chromatography (HSCCC) for further confirmation of their stabilities of G-quadruplex by temperature-dependent circular dichroism (CD). Four compounds were isolated and identified as 2,4-dihydroxybenzoic acid (I), chlorogenic acid (II), caffeic acid (III) and 5-feruloylquinic acid (IV) each by MS and NMR. Finally, compound I, II, III were each proved to be potent G-quadruplex ligands by decreasing the peak intensity in HPLC chromatogram after complexation with G-quadruplex, which stabilize G-quadruplex by 7±2 °C, 10±2 °C, and 3±2 °C respectively, based on CD analyses. However, compound IV showed no G-quadruplex stability. The decrease of peak absorption intensity in HPLC chromatogram is the most important signal to find G-quadruplex ligands. This provides a very promising strategy for fast screening G-quadruplex ligands from natural plant extracts.     

Cyclometalated platinum(II) complexes as topoisomerase IIα poisons

A platinum(II)-based major groove binder [Pt(II)(C^N)(C≡NR)(2)](+) (HC^N = 2-phenylpyridine (phpy), R = 2-naphthyl) was identified as a potent human topoisomerase IIα poison. It stabilizes the covalent TopoIIα-DNA cleavage complex and induces cancer cell death with potency significantly higher than the widely clinically used TopoIIα poison Vp-16.     

Asymmetric total synthesis of ent-(-)-roseophilin: assignment of absolute configuration

An asymmetric total synthesis of ent-(-)-roseophilin (1), the unnatural enantiomer of a novel naturally occurring antitumor antibiotic, is described. The approach enlists a room temperature heterocyclic azadiene inverse electron demand Diels-Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (7) with the optically active enol ether 6 bearing the C23 chiral center followed by a reductive ring contraction reaction for formation of an appropriately functionalized pyrrole ring in a key 1,2,4,5-tetrazine --> 1,2-diazine --> pyrrole reaction sequence. A Grubbs' ring closing metathesis reaction was utilized to close the unusual 13-membered macrocycle prior to a subsequent 5-exo-trig acyl radical-alkene cyclization that was used to introduce the fused cyclopentanone and complete the preparation of the tricylic ansa-bridged azafulvene core 32. Condensation of 32 with 33 under the modified conditions of Tius and Harrington followed by final deprotection provided (22S,23S)-1. Comparison of synthetic (22S,23S)-1 ([alpha](25)(D), CD) with natural 1 established that they were enantiomers and enabled the assignment of the absolute stereochemistry of the natural product as 22R,23R. Surprisingly, ent-(-)-1 was found to be 2-10-fold more potent than natural (+)-1 in cytotoxic assays, providing an unusually rewarding culmination to synthetic efforts that provided the unnatural enantiomer.     

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.     

Benzothiazole-substituted benzofuroquinolinium dye: a selective switch-on fluorescent probe for G-quadruplex

A new switch-on fluorescent probe containing the natural product cryptolepine analogue benzofuroquinolinium moiety (binding scaffold) and a benzothiazole moiety (signalling unit) shows a remarkable fluorescence enhancement selective for the G-quadruplex nucleic acid structure. Binding studies revealed that the highly selective response of the fluorescent probe arises from end-stack binding to G-quadruplex.     

Total synthesis of (-)- and (+)-dysibetaine

Glycidamides 6R and 6S were elaborated to (R,R)- and (S,S)-dysibetaines (1R and 1S) by intramolecular alkylation and functional group modification in 23% overall yield. The absolute stereochemistry of natural dysibetaine was established as S,S by comparison of the optical rotation of the natural product with that of the synthetic materials.     

Cryptolepine Derivatives: Quadruplex-Interactive Agents as Inhibitors of Human Telomerase

Alkaloids are very important natural products. Most of them have biologic activity. Many novel drugs have been developed based on alkaloids, such as camptothecin, taxol, vinblastine. A series of novel cryptolepine derivatives were synthesized (Figure 1). The interaction of cryptolepine derivatives with G-quadruplex (Figure 2) was studied by CD and UV spectra.[1] Most of these compounds can induce the formation of G-quadruplex and stabilize the formed G-quadruplex, resulting in the inhibitory effect on telomerase. Most of these cryptolepine derivatives have potent cytotoxicity in vitro against human tumor cell line.     

Formation and recognition of G-quadruplex in promoter of c-myb oncogene by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) is used to study the formation of G-quadruplex by d(GGAGGAGGAGGA) which locates at the promoter region of c-myb gene. In addition, a natural small molecule, dehydrocorydaline from a Chinese herb, is found to have the highest binding affinity with the G-quadruplex in nine natural small molecules studied, and the binding selectivity of this natural molecule toward the c-myb G-quadruplex with respect to corresponding duplex DNA is significantly higher than that of the broad-spectrum G-quadruplex-ligand TMPyP4. The result from ESI-MS indicates that the gas-phase kinetic stability of the G-quadruplex can be enhanced by binding of dehydrocorydaline. To further investigate the binding properties of dehydrocorydaline to the G-quadruplex, Autodock3 is used to calculate the docked sites and docked energies of small molecules binding to the G-quadruplex and the result shows that the docked energy of dehydrocorydaline is the biggest in the nine small molecules used, consistent with the result from ESI-MS.