Structure and biological evaluation of pyridine-2-carboxamidine copper(II) complex resulting from N'-(4-nitrophenylsulfonyloxy)2-pyridine-carboxamidoxime.

The interaction of Cu(NO3)2·3H2O with the sulfonyl o-pyridine carboxamidoxime N'-(4-nitrophenylsulfonyloxy)picolinimidamide (L) resulted in the mononuclear complex [Cu(L1)2](L2)2 (1), where L1 = pyridine-2-carboxamidine ligand and (L2)- = 4-nitrobenzenesulfonate anion derived from the homolytic cleavage of the NO bond of L. The complex was characterized by diverse techniques including single-crystal X-ray crystallography. From the antimicrobial tests performed, complex 1 seems to be active against gram-negative bacterial strains. The complex binds tightly and reversibly to serum albumins and tightly to calf-thymus DNA via an intercalative mode and also via electrostatic interactions (as expected due to its cationic nature). Additionally, it interacts with (pBluescriptSK(+)) plasmid DNA in a concentration-dependent manner. The results from the present in silico molecular modeling simulations provide useful complementary insights for the elucidation of the mechanism of action of the studied complex at a molecular level. Molecular modeling calculations provide a molecular basis for the understanding of both the impairment of DNA by its binding with the studied complex and the ability of this compound to act as an antibacterial agent, most probably by its activity against DNA-gyrase, as well as for transportation through serum albumins and possible interaction with other protein targets involved in various diseases.

Genotoxic activity of 1,2,3-triazolyl modified furocoumarins and 2,3-dihydrofurocoumarins.

The furocoumarin backbone is a promising platform for chemical modifications aimed at creating new pharmaceutical agents. However, the high level of biological activity of furocoumarins is associated with a number of negative effects. For example, some of the naturally occurring ones and their derivatives can show genotoxic and mutagenic properties as a result of their forming crosslinks with DNA molecules. Therefore, a particularly important area for the chemical modification of natural furocoumarins is to reduce the negative aspects of their bioactivity. By studying a group of 21 compounds-1,2,3-triazolyl modified derivatives of furocoumarin and peucedanin-using the SOS chromotest, the Ames test, and DNA-comet assays, we revealed modifications that can neutralize the structure's genotoxic properties. Theoretical aspects of the interaction of the compound library were studied using molecular modeling and this identified the leading role of the polyaromatic molecular core that takes part in stacking-interactions with the pi-systems of the nitrogenous bases of DNA.

The Intercalation of CORM-2 with Pharmaceutical Clay Montmorillonite (MMT) Aids for Therapeutic Carbon Monoxide Release.

The pharmaceutical clay montmorillonite (MMT) is, for the first time, explored as a carbon monoxide-releasing material (CORMat). MMT consists of silicate double layered structure; its exfoliation feature intercalate the CORM-2 [RuCl(µ-Cl)(CO)3]2 inside the layers to suppress the toxicity of organometallic segment. The infrared spectroscopy (IR) confirmed the existence of ruthenium coordinated carbonyl ligand in MMT layers. The energy-dispersive X-ray spectroscopy (EDX) analysis showed that ruthenium element in this material was about 5%. The scanning electron microscopy (SEM) and transmission electron microscope (TEM) images showed that the layer-structure of MMT has been maintained after loading the ruthenium carbonyl segment. Moreover, the layers have been stretched out, which was confirmed by X-ray diffraction (XRD) analysis. Thermogravimetric (TG) curves with huge weight loss around 100-200 °C were attributed to the CO hot-release of ruthenium carbonyl as well as the loss of the adsorbed solvent molecules and the water molecules between the layers. The CO-liberating properties have been assessed through myoglobin assay. The horse myoglobin test showed that the material could be hydrolyzed to slowly release carbon monoxide in physiological environments. The half-life of CO release was much longer than that of CORM-3, and it has an excellent environmental tolerance and slow release effect.

Intercalating Electron Dyes for TEM Visualization of DNA at the Single-Molecule Level.

Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules by using TEM. However, research into the synthesis and utilization of alternative electron dyes has been limited. Here, we report the synthesis of a novel DNA intercalator molecule, bis-acridine uranyl (BAU). NMR spectroscopy and MS confirmed the validity of the synthetic strategy and gel electrophoresis verified the binding of BAU to DNA. For TEM imaging of DNA, two-dimensional DNA origami nanostructures were used as a robust microscopy test object. By using scanning transmission electron microscopy (STEM) imaging, which is favored over conventional wide-field TEM for improved contrast, and therefore, quantitative image analysis, it is found that the synthesized BAU intercalator can render DNA visible, even at the single-molecule scale. For comparison, other staining compounds with a purported affinity towards DNA, such as dichloroplatinum, cisplatin, osmium tetroxide, and uranyl acetate, have been evaluated. The STEM contrast is discussed in terms of the DNA-dye association constants, number of dye molecules bound per base pair, and the electron-scattering capacity of the metal-containing ligands. These findings pave the way for the future development of electron dyes with specific DNA-binding motifs for high-resolution TEM imaging.

DNA-Hybridization Detection on Si(100) Surfaces Using Light-Activated Electrochemistry: A Comparative Study between Bovine Serum Albumin and Hexaethylene Glycol as Antifouling Layers.

Light can be used to spatially resolve electrochemical measurements on a semiconductor electrode. This phenomenon has been explored to detect DNA hybridization with light-addressable potentiometric sensors and, more recently, with light-addressable amperometric sensors based on organic-monolayer-protected Si(100). Here, a contribution to the field is presented by comparing sensing performances when bovine serum albumin (BSA) and hexaethylene glycol (OEG6) are employed as antifouling layers that resist nonspecific adsorption to the DNA-modified interface on Si(100) devices. What is observed is that both sensors based on BSA or OEG6 initially allow electrochemical distinction among complementary, noncomplementary, and mismatched DNA targets. However, only surfaces based on OEG6 can sustain electroactivity over time. Our results suggest that this relates to accelerated SiO x formation occasioned by BSA proteins adsorbing on monolayer-protected Si(100) surfaces. Therefore, DNA biosensors were analytically explored on low-doped Si(100) electrodes modified on the molecular level with OEG6 as an antifouling layer. First, light-activated electrochemical responses were recorded over a range of complementary DNA target concentrations. A linear semilog relation was obtained from 1.0 × 10-11 to 1.0 × 10-6 mol L-1 with a correlation coefficient of 0.942. Then, measurements with three independent surfaces indicated a relative standard deviation of 4.5%. Finally, selectivity tests were successfully performed in complex samples consisting of a cocktail mixture of four different DNA sequences. Together, these results indicate that reliable and stable light-activated amperometric DNA sensors can be achieved on Si(100) by employing OEG6 as an antifouling layer.

Molecular Recognition of the Hybrid-2 Human Telomeric G-Quadruplex by Epiberberine: Insights into Conversion of Telomeric G-Quadruplex Structures.

Human telomeres can form DNA G-quadruplex (G4), an attractive target for anticancer drugs. Human telomeric G4s bear inherent structure polymorphism, challenging for understanding specific recognition by ligands or proteins. Protoberberines are medicinal natural-products known to stabilize telomeric G4s and inhibit telomerase. Here we report epiberberine (EPI) specifically recognizes the hybrid-2 telomeric G4 predominant in physiologically relevant K+ solution and converts other telomeric G4 forms to hybrid-2, the first such example reported. Our NMR structure in K+ solution shows EPI binding induces extensive rearrangement of the previously disordered 5'-flanking and loop segments to form an unprecedented four-layer binding pocket specific to the hybrid-2 telomeric G4; EPI recruits the (-1) adenine to form a "quasi-triad" intercalated between the external tetrad and a T:T:A triad, capped by a T:T base pair. Our study provides structural basis for small-molecule drug design targeting the human telomeric G4.

Computational DNA binding studies of (-)-epigallocatechin-3-gallate.

The catechin family of molecules that are present in the leaves of green tea has been under investigation since the antioxidant and anti-inflammatory properties of tea were discovered. Among multiple proposed therapeutic targets of these molecules, the direct interaction with nucleic acids has been proposed and experimentally observed but without clear knowledge about the potential binding modes between these ligands and DNA. One of these catechin structures, (-)-epigallocatechin gallate (EGCG), has three aromatic rings that could interact with double-stranded DNA via terminal base-pair stacking, intercalation, or through groove binding. Using enhanced sampling techniques and molecular dynamics simulations, we have found a stable complex between the EGCG ligand and DNA through intercalation of the trihydroxybenzoate aromatic ring and an ApC step. Moreover, we have calculated the absorption spectra of four possible binding modes and compared these to absorption profiles reported in the literature, and explored the possible DNA sequence preference for the EGCG ligand to bind. Our results suggest that an intercalative mode of interaction through the major groove is possible between the EGCG ligands and DNA with apparently very little DNA sequence selectivity.

Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction.

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC50 of 0.82 ± 0.18 µM and 1.3 ± 0.22 µM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.

Anticancer activity of novel amino acid derivative of palladium complex with phendione ligand against of human colon cancer cell line.

The aim of this work is the identification of the structural effect of amino acid-Pd complex on DNA as an intracellular target which was studied using various spectroscopic techniques such as fluorescence, UV-visible and circular dichroism in combination with a molecular docking study. Hence, a novel water-soluble palladium complex, [Pd(phendione)(isopentylglycine)]NO3, has been synthesized and characterized by spectroscopic method. The anticancer activity of complex was investigated against human colon cancer cell line of HCT116 after 24 h of incubation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In addition, this complex was interacted with calf thymus DNA (ct-DNA) via positive cooperative interaction. The fluorescence data indicate that Pd complex is intercalated in DNA. These results were confirmed by circular dichroism spectra. The molecular docking results indicate that docking may be an appropriate method for the prediction and confirmation of experimental results. Complementary molecular docking results may be useful for the determination of the binding mechanism of DNA in pharmaceutical and biophysical studies providing new insight into the novel pharmacology and new solutions in the formulation of advanced oral drug delivery systems. Docking and spectroscopic studies show that new water-soluble Pd complex has anticancer activity and it can bind to DNA via intercalation and groove binding.

Selenium nanoparticles synthesized in aqueous extract of Allium sativum perturbs the structural integrity of Calf thymus DNA through intercalation and groove binding.

Biomedical application of selenium nanoparticles (SeNPs) demands the eco-friendly composite for synthesis of SeNPs. The present study reports an aqueous extract of Allium sativum (AqEAS) plug-up the current need. Modern spectroscopic, microscopic and gravimetric techniques were employed to characterize the synthesized nanoparticles. Characterization studies revealed the formation of crystalline spherical shaped SeNPs. FTIR spectrum brings out the presence of different functional groups in AqEAS, which influence the SeNPs formation and stabilization. Furthermore the different aspects of the interaction between SeNPs and CT-DNA were scrutinized by various spectroscopic and cyclic voltametric studies. The results reveals the intercalation and groove binding mode of interaction of SeNPs with stacked base pair of CT-DNA. The Stern-Volmer quenching constant (KSV) were found to be 7.02×106M-1 (ethidium bromide), 4.22×106 M-1 (acridine orange) and 7.6×106M-1 (Hoechst) indicating strong binding of SeNPs with CT-DNA. The SeNPs - CT-DNA interactions were directly visualized by atomic force microscopy. The present study unveils the cost effective, innocuous, highly stable SeNPs intricate mechanism of DNA interaction, which will be a milestone in DNA targeted chemotherapy.

Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples.

Propidium monoazide treatment to distinguish between live and dead methanogens in pure cultures and environmental samples.

In clinical trials investigating human health and in the analysis of microbial communities in cultures and natural environments, it is a substantial challenge to differentiate between living, potentially active communities and dead cells. The DNA-intercalating dye propidium monoazide (PMA) enables the selective masking of DNA from dead, membrane-compromised cells immediately before DNA extraction. In the present study, we evaluated for the first time a PMA treatment for methanogenic archaea in cultures and particle-rich environmental samples. Using microscopic analyses, we confirmed the applicability of the LIVE/DEAD(®) BacLight™ kit to methanogenic archaea and demonstrated the maintenance of intact cell membranes of methanogens in the presence of PMA. Although strain-specific differences in the efficiency of PMA treatment to methanogenic archaea were observed, we developed an optimal procedure using 130 µM PMA and 5min of photo-activation with blue LED light. The results showed that the effectiveness of the PMA treatment strongly depends on the texture of the sediment/soil: silt and clay-rich sediments represent a challenge at all concentrations, whereas successful suppression of DNA from dead cells with compromised membranes was possible for low particle loads of sandy soil (total suspended solids (TSS)≤200 mg mL(-1)). Conclusively, we present two strategies to overcome the problem of insufficient light activation of PMA caused by the turbidity effect (shielding) in particle-rich environmental samples by (i) dilution of the particle-rich sample and (ii) detachment of the cells and the free DNA from the sediment prior to a PMA treatment. Both strategies promise to be usable options for distinguishing living cells and free DNA in complex environmental samples.

A new DNA-intercalative cytotoxic allylic xanthone from Swertia corymbosa.

Phytochemical investigation of the CHCl3 fraction of Swertia corymbosa resulted in the isolation of a new 3-allyl-2,8-dihydroxy-1,6-dimethoxy-9H-xanthen-9-one (1), along with four known xanthones, gentiacaulein (3), norswertianin (4), 1,3,6,8-tetrahydroxyxanthone (5), and 1,3-dihydroxyxanthone (6). Structure of compound 1 was elucidated with the aid of IR, UV, NMR, and MS data, and chemical transformation via new allyloxy xanthone derivative (2). Compounds 1-6 exhibited various levels of antioxidant and anti-α-glucosidase activities. Absorption and fluorescence spectroscopic studies on 1-6 indicated that these compounds could interact with calf thymus DNA (CT-DNA) through intercalation and with bovine serum albumin (BSA) in a static quenching process. Compound 1 was found to be significantly cytotoxic against human cancer cell lines HeLa, HCT116, and AGS, and weakly active against normal NIH 3T3 cell line.

An analysis method for simultaneous screening of deoxyribonucleic acid-binding active compounds and investigating their mechanisms by ultra-fast liquid chromatography tandem mass spectrometry coupled with fluorescence detection technology.

DNA has been known as the cellular target for many cytotoxic anticancer agents over the years. Discovering DNA-binding compounds has become an active research area, while various DNA-binding mechanisms make the drug discovery even more difficult. In this article, we present a novel analysis method to rapidly identify specific DNA-binding compounds from Pyrrosia lingua (Thunb.) using DNA-dual-fluorescent probes, ethidium bromide and Hoechst 33258, with the technology of ultra-fast liquid chromatography-diode array detector-tandem mass spectrometry and dual-wavelength fluorescence detector (UFLC-DAD-MS(n)-DFLD). Sixty-two compounds were identified, of which 22 were found to be active in DNA-binding. After investigation of their dose-response behaviors and structure-activity relationships, chlorogenic acids and flavonoid glycosides were found to be DNA-binders via both minor groove-binding and intercalation modes. The precision, reproducibility and stability of this method were validated by vitexin. The established system was sensitive, precise, and reliable to be used for both screening of DNA-binding compounds and investigating of their mechanisms.

Interaction of YOYO-1 with guanine-rich DNA.

The oxazole homodimer YOYO-1 has served as a valuable tool for the detection and quantification of nucleic acids. While the base specificity and selectivity of binding of YOYO-1 has been researched to some extent, the effect of unorthodox nucleic acid conformations on dye binding has received relatively less attention. In this work, we attempt to correlate the quadruplex-forming ability of G-rich sequences with binding of YOYO-1. Oligonucleotides differing in the number of tandem G repeats, total length, and length of loop sequence were evaluated for their ability to form quadruplexes in presence of sodium (Na(+)) or potassium (K(+)) ions. The fluorescence behavior of YOYO-1 upon binding such G-rich sequences was also ascertained. A distinct correlation was observed between the strength and propensity of quadruplex formation, and the affinity of YOYO-1 to bind such sequences. Specifically, as exemplified by the oligonucleotides 5'-G4T2G4-3' and 5'-G3TG3TG3-3', sequences possessing longer G-rich regions and shorter loop sequences formed stronger quadruplexes in presence of K(+) which translated to weaker binding of YOYO-1. The dependence of binding of YOYO-1 on sequence and structural features of G-rich DNA has not been explored previously and such studies are expected to aid in more effective interpretation of applications involving the fluorophore.

Synthesis and biological evaluation of a class of quinolone triazoles as potential antimicrobial agents and their interactions with calf thymus DNA.

A novel series of quinolone triazoles were synthesized and characterized by IR, NMR, MS and HRMS spectra. All the newly prepared compounds were screened for their antimicrobial activities against seven bacteria and four fungi. Bioactive assay manifested that most of new compounds exhibited good or even stronger antibacterial and antifungal activities against the tested strains including multi-drug resistant MRSA in comparison with reference drugs Norfloxacin, Chloromycin and Fluconazole. The preliminary interactive investigations of compound 6b with calf thymus DNA by fluorescence and UV-vis spectroscopic methods revealed that compound 6b could effectively intercalate DNA to form compound 6b-DNA complex which might block DNA replication and thus exert its antimicrobial activities.

Electrochemiluminescent biosensor of ATP using tetrahedron structured DNA and a functional oligonucleotide for Ru(phen)3(2+) intercalation and target identification.

Restricted target accessibility and surface-induced perturbation of the aptamer structure are the main limitations in single-stranded DNA aptamer-based electrochemical sensors. Chemical labeling of the aptamer with a probe at the end of aptamer is inefficient and time-consuming. In this work, tetrahedron-structured DNA (ts-DNA) and a functionalized oligonucleotide (FO) were used to develop an electrochemiluminescence (ECL) aptasensor with adenosine triphosphate (ATP) as a model target. The ts-DNA was formed with three thiolated oligonucleotides and one oligonucleotide containing anti-ATP aptamer. The FO contained a complementary strand to the anti-ATP aptamer and an intermolecular duplex for Ru(phen)3(2+) intercalation. After the ts-DNA was immobilized on the electrode surface through gold-thiol interactions, hybridization between the anti-ATP aptamer and its complementary strand introduced the intercalated Ru(phen)3(2+) to the electrode. ECL emission from Ru(phen)3(2+) was observed with tripropylamine as a co-reactant. Once ATP reacted with its aptamer, the aptamer-complimentary strand duplex dissociated and the intermolecular duplex containing Ru(phen)3(2+) was released. The difference in emission before and after reaction with ATP was used to quantify ATP with a detection limit of 0.2nM. The ts-DNA increased the sensitivity compared to conventional methods, and the intercalation strategy avoided a complex chemical labeling procedure.

Membrane perturbations induced by new analogs of neocryptolepine.

Indoloquinoline alkaloids represent an important class of antimalarial, antibacterial and antiviral compounds. Indolo[2,3-b]quinolines are a family of DNA intercalators and inhibitors of topoisomerase II, synthetic analogs of neocryptolepine, an alkaloid traditionally used in African folk medicine. These cytotoxic substances are promising anticancer agents. Active representatives of indolo[2,3-b]quinolines affect model and natural membranes. The distinct structure and hydrophobicity of the compounds leads to marked differences in the disturbing effects on membrane organization and function. Our results also indicated a strong relationship between the presence of the chain and the Poct of the molecule as well as the capacity for incorporation into carboxyfluorescein-trapped liposomes in the 0.02-0.06 mM range. Moreover, a correlation between binding to neutral dimyristoylphosphatidylcholine (DMPC) or negative charged dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DMPC:DMPG, 9:1 w/w) liposomes, as well as to erythrocyte ghosts and pKa, was also found. All the compounds cause hemolysis in isotonic conditions with concentration causing 50% hemolysis (HC50) in the 0.12-0.88 mM range. The concentration-dependent inhibitory effect of the tested agents on erythrocyte ghosts' acetylcholinesterase activity was also studied.

Quantification of dye-mediated photodamage during single-molecule DNA imaging.

Single-molecule fluorescence imaging of DNA-binding proteins has enabled detailed investigations of their interactions. However, the intercalating dyes used to visually locate DNA molecules have the undesirable effect of photochemically damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single-strand breaks (SSBs), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSBs on DNA molecules by the dye YOYO-1 using complementary single-molecule imaging and gel electrophoresis-based damage assays. The single-molecule assay imaged hydrodynamically elongated lambda DNA, enabling the real-time detection of double-strand breaks (DSBs). The gel assay, which used supercoiled plasmid DNA, was sensitive to both SSBs and DSBs. This enabled the quantification of SSBs that precede DSB formation. Using the parameters determined from the gel damage assay, we applied a model of stochastic DNA damage to the time-resolved DNA breakage data, extracting the rates of single-strand breakage at two dye staining ratios and measuring the damage reduction from the radical scavengers ascorbic acid and ß-mercaptoethanol. These results enable the estimation of the number of SSBs that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.

Synthesis, characterization, nucleic acid binding, and cytotoxic activity of a Ru(II) polypyridyl complex.

The binding properties of [Ru(bpy)(2)(H(2)IIP)](2+) (1) {bpy=2,2'-bipyridine, H(2)IIP=2-(indole-3-yl)-imidazolo[4,5-f][1,10]phenanthroline} with calf thymus DNA (CT-DNA) and yeast tRNA have been investigated comparatively by different spectroscopic and viscosity measurements. The results suggest that the affinity of complex 1 binding with yeast tRNA is stronger than that of complex 1 binding with CT-DNA, and complex 1 is a better enantioselective binder to yeast tRNA than to CT-DNA. The toxicity of complex 1 was concentration dependent, and HL-60 cells are more sensitive to complex 1 than Hep-G2 cells; complex 1 could induce Hep-G2 cell apoptosis.