G4LDB 2.2: a database for discovering and studying G-quadruplex and i-Motif ligands.

Noncanonical nucleic acid structures, such as G-quadruplex (G4) and i-Motif (iM), have attracted increasing research interests because of their unique structural and binding properties, as well as their important biological activities. To date, thousands of small molecules that bind to varying G4/iM structures have been designed, synthesized and tested for diverse chemical and biological uses. Because of the huge potential and increasing research interests on G4-targeting ligands, we launched the first G4 ligand database G4LDB in 2013. Here, we report a new version, termed G4LDB 2.2 (http://www.g4ldb.com), with upgrades in both content and function. Currently, G4LDB2.2 contains >3200 G4/iM ligands, ∼28 500 activity entries and 79 G4-ligand docking models. In addition to G4 ligand library, we have also added a brand new iM ligand library to G4LDB 2.2, providing a comprehensive view of quadruplex nucleic acids. To further enhance user experience, we have also redesigned the user interface and optimized the database structure and retrieval mechanism. With these improvements, we anticipate that G4LDB 2.2 will serve as a comprehensive resource and useful research toolkit for researchers across wide scientific communities and accelerate discovering and validating better binders and drug candidates.

Design and Screening of Fluorescent Probes Based upon Hemicyanine Dyes to Sensitively Respond to HSO3- in Living Cells.

SO2, a gas signaling molecule, can be produced endogenously in mitochondria. Its hydrolysate, HSO3-, plays a key role in food preservation, cardiovascular relaxation, and other fields, suggesting that it is important to achieve its detection. Here, based on the Michael addition mechanism, four hemicyanine dye fluorescent probes (ETN, ETB, STB, and EIB) were designed and synthesized for responding to HSO3-. We evaluated the reaction ability of different probes with HSO3- and tried to explain the reasons for the significantly different response effects between probes and HSO3- according to the structure-activity relationship. The influence of different substituents of probes on the properties of mitochondria-targeting was also discussed. Finally, we screened out ETN as the optimal HSO3- probe due to its high sensitivity, rapid reactivity, and good mitochondria-targeting, and it could sensitively respond to HSO3- in living cells. The LODs of ETN for HSO3- were calculated by both absorption and fluorescence methods, respectively, which were 2.727 and 0.823 µM. Our work provided valuable references for designing strategies and potential tools for response to SO2 derivatives in biosystems.

Selective recognition of DNA parallel G-quadruplexes by 3,8a-disubstituted indolizinones.

Owing to its potential biological relevance, DNA G-quadruplex has been considered as a prospective anti-cancer target. Some known G-quadruplex-interactive N-containing compounds with low cytotoxicity have become prospective anticancer drugs. Here we reported a new type of N-containing alkaloids 3,8a-disubstituted indolizinones, and investigated their substituent effects at 3- and 8a-positions in targeting to DNA c-myc G-quadruplex. And then we used 3-naphtyl-8a-(pyridin-2-yl) substrate I8 as an example, and investigated its ability in targeting to DNA parallel G-quadruplexes in vitro.

Stabilizing G-quadruplex DNA by methylazacalix[n]pyridine through shape-complementary interaction.

It is found that G-quadruplexes have important functions in biological systems, such as gene expression. Molecules which can stabilize the G-quadruplex structure may have potential application in regulating the expression of gene. A series of methylazacalix[n]pyridine (n=4, 6, 7, 8, 9) has been tested to stabilize the intermolecular human telomeric G-quadruplex (T12 and H12), intramolecular TBA, c-kit and bcl-2 G-quadruplex by CD denaturation experiments. The results showed that only methylazacalix[6]pyridine (MACP6) can stabilize the intermolecular G-quadruplex formed from the 12bp human telomere. Further studies evidenced that the shape-complementary binding mode was what contributed to the interaction between MACP6 and T12 G-quadruplex.

G4LDB: a database for discovering and studying G-quadruplex ligands.

The G-quadruplex ligands database (G4LDB, http://www.g4ldb.org) provides a unique collection of reported G-quadruplex ligands to streamline ligand/drug discovery targeting G-quadruplexes. G-quadruplexes are guanine-rich nucleic acid sequences in human telomeres and gene promoter regions. There is a growing recognition for their profound roles in a wide spectrum of diseases, such as cancer, diabetes and cardiovascular disease. Ligands that affect the structure and activity of G-quadruplexes can shed light on the search for G-quadruplex-targeting drugs. Therefore, we built the G4LDB to (i) compile a data set covering various physical properties and 3D structure of G-quadruplex ligands; (ii) provide Web-based tools for G-quadruplex ligand design; and (iii) to facilitate the discovery of novel therapeutic and diagnostic agents targeting G-quadruplexes. G4LDB currently contains >800 G-quadruplex ligands with ∼4000 activity records, which, to our knowledge, is the most extensive collection of its kind. It offers a user friendly interface that can meet a variety of data inquiries from researchers. For example, ligands can be searched for by name, molecular properties, structures, ligand activities and so on. Building on the reported data, the database also provides an online ligand design module that can predict ligand binding affinity in real time.

Study on the interaction of a cyanine dye with human serum transferrin.

Complexation between the primary carrier of ligands in blood plasma, human serum transferrin (Tf), and a cyanine dye, 3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-phenyl-thiacarbocyanine-triethylam monium salt (PTC) was investigated using fluorescence spectra, UV/Vis absorption spectra, synchronous fluorescence spectra, circular dichroism (CD) and molecular dynamic docking. The experimental results demonstrate that the formation of PTC-Tf complex is stabilized by van der Waal's interactions and hydrogen bonds, and the binding constants were found to be 8.55 × 10(6), 8.19 × 10(6) and 1.75 × 10(4) M(-1). Moreover, fluorescence experiments prove that the operational mechanism for the fluorescence quenching is static quenching and non-radiative energy transfer. Structural investigation of the PTC-Tf complexes via synchronous fluorescence spectra and CD showed that the structure of Tf became more stable with a major increase in the α-helix content and increased polarity around the tryptophan residues after PTC binding. In addition, molecular modeling highlights the residues located in the N-lobe, which retain high affinity for PTC. The mode of action of the PTC-Tf complex is illustrated by these results, and may provide an effective pathway for the transport and targeted delivery of antitumor agents.

[Investigation on the cyanine dyes supramolecular assembly and chiral inducement by fulvic acid].

Using cyanine dyes supramolecular as molecular probes to mark FA has important significance in life sciences and pharmaceutical chemistry, which can detect FA as drug efficacy mechanism and the change in physiological activity. In the present paper, we investigated supramolecular assembly and chiral inducement of cyanine dyes template by FA with absorption and circular dichroism (CD) spectra. The result suggests that FA can induce cyanine dyes from J-aggregation to monomer along with different colors change and has strong affinity with cyanine dye monomer. The template of FA not only can translate the chirality of MTC H-aggregation to other two states, but also induce ETC J-aggregation to molecular rearrangement and form left-handed helix of J-aggregates. Besides, the association of PTC with FA, i. e. binding to FA gave rise to the J-aggregation CD signals. Meanwhile, it was inferred that the meso substituent of cyanine dyes play an important role in the interaction between FA and the J-aggregation: the smaller the meso substituent, the higher the affinity interacted with FA. Clearly, the binding abilities between cyanine dyes and FA follow the order of MTC>ETC>PTC. These results support that the cyanine dyes supramolecular aggregates can be used as a kind of excellent molecular probes for specific recognition of FA and achieve the effect of visual inspection.

The effect of the skeleton structure of flavanone and flavonoid on interaction with transferrin.

Transferrin has been exploited as a potential drug carrier for targeted drug delivery into cancer cells, which express high levels of transferrin receptors. In the present study, we identified specific structural features in flavonoids that were critical for binding to transferrin. Flavanone naringenin and flavonoid apigenin, two flavonoids with characteristic flavonoid core structures were selected for the study of the effects of C2-C3 single bond in the C-ring on transferrin binding. We determined the binding affinities by fluorescence quenching experiments and investigated the binding modes by CD spectra and molecular modeling. Our results demonstrated that naringenin bound transferrin with an affinity almost 100 times higher than that of apigenin attributed to its higher structural flexibility and lower acidity compared with apigenin. Our docking study showed that naringenin had stronger van der Waals interactions with transferrin, which was believed to contribute to its higher binding affinity. We also found that naringenin-binding induced greater increase in the α-helix content in transferrin than apigenin, suggesting that transferrin became more compact upon association with naringenin. Our study demonstrated that naringenin was a ligand for transferrin with good affinity. The results reported herein can facilitate the design and development of drugs that bind transferrin with high affinity.

Formation of an intramolecular G-quadruplex of human telomere induced by poly(L-lysine) under salt-deficient conditions.

A single-stranded G-tract human telomere DNA sequence is able to fold into intramolecular G-quadruplex structures which may be important for a number of biological processes and disease-related mechanisms. Poly(L-lysine) (PLL) polymer is linear polypeptides with lysine as the repeat unit and has been employed as a gene carrier in achieving targeted delivery of DNA to cancer cells. To explore the influence of PLL on the conformation of Hum24 DNA, we have investigated the interaction of PLL with Hum24 by biophysical methods, mainly CD, ESI-MS, and polyacrylamide gel electrophoresis for the first time. The CD data have shown that PLL can induce single-stranded Hum24 to form an intramolecular parallel G-quadruplex structure, further confirmed by ESI-MS analysis and gel electrophoresis results. The formation of an intramolecular G-quadruplex is strongly dependent on the Hum24/PLL molar ratios and the length of both the polypeptides and oligonucleotide. Such phenomena may be interpreted by electrostatically attracting negative-charged Hum24 by positive-charged PLL which facilitates the close contact between the guanines and formation of hydrogen bonding, thus leading the final shape of a G-quadruplex structure.

[Anti-tumor activity of Erythrina variegata L. extract and its mechanism of action].

The anti-tumor activities and mechanism of Erythrina variegata L. extract were investigated. Firstly, the MTT method was used to evaluate the inhibitory activity of the Erythrina variegata L. extract on proliferation of cancer cell lines. Moreover, in order to determine its anti-tumor effect in vivo, the Lewis lung cancer mice model was established. By comparing the relative tumor proliferation rates, growth curves, inhibition rates of different groups, the anti-tumor effect was evaluated. Furthermore, the anti-tumor mechanism of Erythrina variegata L. extract was studied by using G-quadruplex stability experiment. In the in vitro anti-liver cancer experiment, the Erythrina variegata L. extract has shown obvious anti-tumor effect on various tumor cells. And in the in vivo experiment, it exhibited significant anti-tumor effect. Besides, from the result of G-quadruplex stability experiment, we can see that the quadruplex structure show increasing T(m) values with increasing amounts of Erythrina variegata L. extract.

Spontaneous formation and reversible transformation between achiral J- and chiral H-aggregates of cyanine dye MTC.

Chirality at a supramolecular level is currently attracting great attention attributed to rapid developments in supramolecular chemistry. Herein, we report a new type of chiral self-assembly based on the cyanine dye MTC. The chiral H-aggregates of MTC could form spontaneously from achiral J-aggregates, and could return back to achiral J-aggregates in high concentration on increasing the solution temperature.

Ethyl-substitutive Thioflavin T as a highly-specific fluorescence probe for detecting G-quadruplex structure.

G-quadruplex has attracted considerable attention due to their prevalent distribution in functional genomic regions and transcripts, which can importantly influence biological processes such as regulation of telomere maintenance, gene transcription and gene translation. Artificial receptor study has been developed for accurate identification of G-quadruplex from DNA species, since it is important for the G-quadruplex related basic research, clinical diagnosis, and therapy. Herein, fluorescent dye ThT-E, a derivative of the known fluorescence probe Thioflavin T (ThT), was designed and synthesized to effectively differentiate various G-quadruplex structures from other nucleic acid forms. Compared with methyl groups in ThT, three ethyl groups were introduced to ThT-E, which leads to strengthened affinity, selectivity and little inducing effect on the G-quadruplex formation. More importantly, ThT-E could be served as a visual tool to directly differentiate G-quadruplex solution even with naked eyes under illumination of ultraviolet light. Thus, this probe reported herein may hold great promise for high-throughput assay to screen G-quadruplex, which may widely apply to G-quadruplex-based potential diagnosis and therapy.

Probing paeonol-pluronic polymer interactions by 1H NMR spectroscopy.

By using a combination of 1H NMR spectroscopy, two-dimensional heteronuclear single-quantum coherence-resolved (1)H{(13)C} and homonuclear rotating-frame Overhauser enhancement NMR correlation experiments with diffusion ordered spectroscopy (DOSY), the location and distribution of a hydrophobic drug, paeonol, have been established with respect to the methyl groups of the poly(ethylene oxide)-poly(propylene oxide) -poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer. The interaction between them is adjustable according to the different temperature-dependent hydrophilicities or hydrophobicities of the triblock copolymer components. On the other hand, such interactions influence the self-assembly properties of the block copolymer amphiphiles in solution. The amount of anhydrous methyl groups of PPO segments shows an increase with increasing paeonol concentration. It was also demonstrated that the shell-crosslinking of the Pluronic polymer has an effect in increasing the amount of anhydrous methyl groups and thus increasing the hydrophobicity of Pluronic micelles. This might be the deeper reason underlying the increase in drug-loading capacity and prolongation in release time of Pluronic micelles for drug delivery after the shell-crosslinking. Changes in self-diffusion coefficients of paeonol with varying copolymer concentrations and types were also determined by the diffusion-based NMR DOSY technique, and values of K(a), DeltaG, and n were calculated.

Interaction of urea with pluronic block copolymers by 1H NMR spectroscopy.

Solution 1H NMR techniques were used to characterize the interaction of urea with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers. The urea was established to interact selectively with the PEO blocks of the block copolymer, and the interaction sites were found not to change with increasing temperature. Such interactions influence the self-assembly properties of the block copolymer in solution by increasing the hydration of the block copolymers and stabilizing the gauche conformation of the PPO chain. Therefore, urea increases the critical micellization temperature (CMT) values of PEO-PPO-PEO copolymers, and the effect of urea on the CMT is more pronounced for copolymers with higher PEO contents and lower for those with increased contents of PPO segments.

Oil-induced aggregation of block copolymer in aqueous solution.

The oil-induced aggregation behavior of PEO-PPO-PEO Pluronic P84 [(EO)19(PO)39(EO)19] in aqueous solutions has been systematically investigated by 1H NMR spectroscopy, freeze-fracture transmission electron microscopy (FF-TEM), and dynamic light scattering (DLS). The critical micellization temperature (CMT) for P84 in the presence of oils decreases with increasing oil concentration. The effectiveness of various oils in decreasing the CMT of block copolymer follows the order m-xylene (C(8)H(10)) > toluene (C(7)H(8)) > benzene (C(6)H(6)) > n-octane (C(8)H(18)) > n-hexane (C(6)H(14)) approximately cyclohexane (C(6)H(12)). It was found that the amount of anhydrous PO methyl groups increases whereas the amount of hydrated PO methyl groups decreases upon the addition of oils. At low oil concentration, the oil molecules are entrapped by the micellar core, but as the oil concentration increases above a certain value, the micellar core swells significantly as a result of the penetrated oil molecules, and much larger aggregates are formed. Intermolecular rotating-frame nuclear Overhauser effect (ROE) measurements between P84 and benzene were performed at 10 and 40 degrees C. The specific interaction between benzene and the methyl groups of PPO was determined, and it was observed that the interaction site remained unchanged as the temperature was increased.

G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch.

Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and (1)H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition.

Isolation of anti-tumor compounds from the stem bark of Zanthoxylum ailanthoides Sieb. & Zucc. by silica gel column and counter-current chromatography.

Silica gel column chromatography combined with high performance counter-current chromatography (HPCCC) was employed for the separation of potential anti-tumor compounds from a petroleum ether fraction of a crude extract of Zanthoxylum ailanthoides Sieb. & Zucc. This traditional Chinese medicine was recently found to display high inhibitory activity against A-549 human cancer cells in vitro and Lewis lung cancer in vivo. A 75% aqueous ethanol extract of the stem bark of Z. ailanthoides was fractionated with petroleum ether, ethyl acetate and n-butanol. In this paper, the petroleum ether fraction was pre-separated by silica gel column chromatography with a petroleum ether-ethyl acetate gradient. Two fractions were further separated and purified by HPCCC using n-hexane-ethyl acetate-methanol-water (3:1:2:1, v/v) and petroleum-ethyl acetate-methanol-water (8:6:7:7, v/v). Finally, coumarins and lignans including luvangetin, xanthyletin, hinokinin and asarinin were isolated and identified by MS, (1)H and (13)C NMR. In total, 56mg of xanthyletin (1), 140mg of hinokinin (2), 850mg of luvangetin (3) and 74mg of asarinin (4) were obtained from approximately 50g of petroleum ether extract, in 96.0%, 94.0%, 99.0% and 94.0% purity, respectively, as determined by HPLC. The separation method proved to be efficient, especially for those minor components.

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.

Screening α-glucosidase inhibitors from mulberry extracts via DOSY and relaxation-edited NNR.

Inhibition of the α-glucosidase activity is a therapeutic approach for diabetes. In this study, an effective strategy for screening α-glucosidase inhibitors based on Nuclear magnetic resonance (NMR) techniques was developed to screen and identify α-glucosidase inhibitors from Mulberry leaf extract. As a result, deoxynojirimycin, as a potential α-glucosidase inhibitor, was found. The study suggested that our strategy was a powerful tool for screening and identification of α-glucosidase inhibitors in complex samples. Furthermore the interaction between α-glucosidase and its inhibitor was studied by NMR.

Regulating the Conformation of Methylazacalix[6]pyridine by Oligonucleotide BCL-2 2345 and Its Recognition of Hydroxymethylpiperazinofullerene.

Calixaromatics have attracted much attention on molecular recognition owing to their flexible conformations, cavity structures, versatile recognition properties, and functions. However, conformational control of calixaromatics is still a challenging topic in the field of calixaromatics. Therefore, we explore the possibility to control the chirality of achiral calixaromatics, methylazacalix[6]pyridine (abbreviated as MACP6), by templating of DNA. We have found that MACP6 with opposite chirality can be achieved by controlling the secondary structure of bcl-2 2345 DNA. Furthermore, MACP6 with different chirality has been used to recognize fullerene derivatives in aqueous solution. Our results have provided a possible approach to construct chiral calixaromatics.