Preferential Binding of a Red Emissive Julolidine Derivative to a Promoter G-Quadruplex.

The therapeutic potential of G-quadruplexes has increased significantly with the growing understanding of their functional roles in pathogens apart from human diseases such as cancer. Here, we report the synthesis of three julolidine-based molecules and their binding to nucleic acids. Among the synthesized molecules, compound 1 exhibited red emissive fluorescence with a distinct preference for Pu22 G-quadruplex. The binding of compound 1 to Pu22 G-quadruplex, initially identified through a fluorescence-based screening, was further confirmed by UV-vis, fluorescence spectroscopy, and circular dichroism-based experiments. Thermal denaturation of compound 1 in the presence of Pu22 G-quadruplex revealed a concentration-dependent stabilization (~10.0 °C at 1 : 3 stoichiometry). Fluorescence-based experiments revealed 1 : 1 stoichiometry of the interaction and an association constant (Ka ) of 5.67×106  M-1 . CD experiments displayed that the parallel conformation of the G-quadruplex was retained on compound 1's binding and signs of higher order binding/complex formation were observed at high compound 1 to DNA ratio. Molecular docking studies revealed the dominance of stacking and van der Waals interactions in the molecular recognition which was aided by some close-distance interactions involving the quinolinium nitrogen atom.

Nile Blue: A Red-Emissive Fluorescent Dye That Displays Differential Self-Assembly and Binding to G-Quadruplexes.

Nile Blue (NB) is a red-emissive dye that is well-known for imaging and staining applications. In this work, we describe the interaction of NB with various types of G-quadruplexes belonging to different topologies, molecularities, and conformations. Using spectroscopic techniques, we have determined the preferential binding of NB to c-Myc G-quadruplex and the other aspects of its binding. Concentration- and temperature-dependent studies showed that NB exists in a dynamic equilibrium between monomeric and H-aggregated states, which could be modulated by the addition of external agents such as anionic surfactants. NB displayed differential self-assembly with different types of G-quadruplex and duplex DNAs modulating its dynamic equilibrium between the monomeric and H-aggregated states. Fluorescence-based displacement studies revealed a 1:1 binding stoichiometry upon interaction with c-Myc G-quadruplex and an association constant of Kapp = 6.7 × 106 M-1. Circular dichroism studies indicated that NB does not cause changes in the overall conformation of either G-quadruplexes or duplexes; however, it does indicate nucleic acid-dependent self-assembly at higher concentrations. Heat capacity measurement showed a more negative change when compared to that in DNA duplex, indicating more burial of the polar surface area by NB to the G-quadruplex host.

Parallel G-quadruplex recognition by neomycin.

G-quadruplex-forming nucleic acids have evolved to have applications in biology, drug design, sensing, and nanotechnology, to name a few. Together with the structural understanding, several attempts have been made to discover and design new classes of chemical agents that target these structures in the hope of using them as future therapeutics. Here, we report the binding of aminoglycosides, in particular neomycin, to parallel G-quadruplexes that exist as G-quadruplex monomers, dimers, or compounds that have the propensity to form dimeric G-quadruplex structures. Using a combination of calorimetric and spectroscopic studies, we show that neomycin binds to the parallel G-quadruplex with affinities in the range of Ka ∼ 105-108 M-1, which depends on the base composition, ability to form dimeric G-quadruplex structures, salt, and pH of the buffer used. At pH 7.0, the binding of neomycin was found to be electrostatically driven potentially through the formation of ion pairs formed with the quadruplex. Lowering the pH resulted in neomycin's association constants in the range of Ka ∼ 106-107 M-1 in a salt dependent manner. Circular dichroism (CD) studies showed that neomycin's binding does not cause a change in the parallel conformation of the G-quadruplex, yet some binding-induced changes in the intensity of the CD signals were seen. A comparative binding study of neomycin and paromomycin using d(UG4T) showed paromomycin binding to be much weaker than neomycin, highlighting the importance of ring I in the recognition process. In toto, our results expanded the binding landscape of aminoglycosides where parallel G-quadruplexes have been discovered as one of the high-affinity sites. These results may offer a new understanding of some of the undesirable functions of aminoglycosides and help in the design of aminoglycoside-based G-quadruplex binders of high affinity.

Effect of the Standardized ZnO/ZnO-GO Filter Element Substrate driven Advanced Oxidation Process on Textile Industry Effluent Stream: Detailed Analysis of Photocatalytic Degradation Kinetics.

A simple process of synthesizing coated filter element substrates (FES) containing zinc oxide (ZnO) nanorods and ZnO graphene-oxide nanocomposite for a pilot-scale industrial dye-effluent treatment plant is proposed. This work reports a detailed analysis of the photocatalysis mechanism on real industrial effluent streams containing a mixture of dyes. The analysis is very relevant for conducting advanced oxidation process-assisted effluent remediation at a field-level treatment operation. Estimation of the dye concentration shows nearly complete (≥98%) degradation from an initial dye sample concentration. A detailed study for the analysis of the initial reactive dyes and their degradation products was performed for quantification and identification of the degradation products through various spectral techniques. A design of the remediation mechanism through degradation pathways is proposed for characterizing the organic compounds in the degraded dye products. A regeneration and reusability study was performed on the FES presenting the durability of the FES-designed synthesis process originally for 11 cycles and regenerated FES for six cycles for achieving a threshold of 60% degradation efficiency. The experimental results demonstrate the efficacy of FES through the designed immobilized approach for the complete remediation of textile dye effluents for a 4 h treatment plant process and the consistent operability of the FES for the combined dye wastewater treatment operations.

Promoter G-Quadruplex Binding Styryl Benzothiazolium Derivative for Applications in Ligand Affinity Ranking and as Ethidium Bromide Substitute in Gel Staining.

Fluorescent compounds that can preferentially interact with certain nucleic acids are of great importance in new drug discovery in a multitude of functions including fluorescence-based displacement assays and gel staining. Here, we report the discovery of an orange emissive styryl-benzothiazolium derivative (compound 4) which interacts preferentially with Pu22 G-quadruplex DNA among a pool of nucleic acid structures containing G-quadruplex, duplex, and single-stranded DNA structures as well as RNA structures. Fluorescence-based binding analysis revealed that compound 4 interacts with Pu22 G-quadruplex DNA in a 1:1 DNA to ligand binding stoichiometry. The association constant (Ka) for this interaction was found to be 1.12 (±0.15) × 106 M-1. Circular dichroism studies showed that the binding of the probe does not cause changes in the overall parallel G-quadruplex conformation; however, signs of higher-order complex formation were seen in the form of exciton splitting in the chromophore absorption region. UV-visible spectroscopy studies confirmed the stacking nature of the interaction of the fluorescent probe with the G-quadruplex which was further complemented by heat capacity measurement studies. Finally, we have shown that this fluorescent probe can be used toward G-quadruplex-based fluorescence displacement assays for ligand affinity ranking and as a substitute for ethidium bromide in gel staining.

Highly Selective and Sensitive Detection of Nitrite Ion by an Unusual Nitration of a Fluorescent Benzimidazole.

Nitrite (NO2-) is a physiologically significant anion having implications for cellular signaling. Here we report our serendipitous discovery of highly selective fluorescence-based nitrite sensing using a benzimidazole which stems from hitherto-unknown direct nitration of a benzimidazole using sodium nitrite. Using one- and two-dimensional NMR techniques, we elucidate the chemical structures of the new nitrated benzimidazoles and show differences in the nitration products using conventional nitration with nitric acid. We also show its utility in robust sensing of nitrite-containing samples.

Fragment-Based Design of Small Molecules to Study DNA Minor Groove Recognition.

DNA-protein interactions are ubiquitous in cellular processes. Impeding unwanted nucleic acid interactions and protein recognition have therapeutic implications. Therefore, new chemical scaffolds and studies related to their structural basis of nucleic acid recognition are essential for developing high-affinity DNA binders. In this study, we have employed a fragment-based strategy to design and synthesize benzimidazole-guanidinium hybrid compounds and study the individual fragment's role in imparting selectivity and specificity in DNA recognition. The fragments were extensively studied using thermal denaturation, circular dichroism, UV-vis absorption spectroscopy, and molecular docking techniques. The results indicate an interdependent role of the benzimidazole core, polar ends, and the DNA composition in imparting sequence-selective binding of the benzimidazole-guanidinium hybrid compounds in the DNA minor groove. Circular dichroism and molecular docking studies indicated minor groove binding analogous to classical minor groove binders such as DAPI and Hoechst 33258. Thermal denaturation studies indicated that the best binder (compound 8) gave similar thermal stabilization to B-DNA as given by DAPI.

Preferential Recognition of Human Telomeric G-Quadruplex DNA by a Red-Emissive Molecular Rotor.

The development of new fluorescent molecules for the recognition of specific G-quadruplex DNA structures has attracted wide attention due to their diverse roles in drug design, sensing, and cellular probing. In this work, we report the discovery of a red-emissive styryl quinolinium-based molecular rotor (compound 1), which recognizes human telomeric G-quadruplex with a distinct preference over DNA duplexes. Optical spectroscopy (UV-vis and circular dichroism)-based experiments indicated discernible interaction of compound 1 with the human telomeric DNA G-quadruplex with features of stacking interactions. Fluorescence-based Job's plot revealed a 1:1 binding stoichiometry between compound 1 and the human telomeric DNA G-quadruplex, and subsequent titration experiments showed micromolar affinities (Ka = 0.51 × 106 M-1). Molecular docking experiments showed interactions of compound 1 in the grooves of the quadruplex. Finally, we provide the application of compound 1 as a reporter molecule in the fluorescence displacement experiments, which showed its ability to act as a fluorescent probe compatible with ligands having aromatic cores.

Oxidation state of graphene oxide nanosheets drives their interaction with proteins: A case of bovine serum albumin.

In the present study, we explored the interaction of bovine serum albumin (BSA) with oxidized graphene oxide (GO) nanosheets. Nanosheets, synthesized with 4, 6, 8, 10 and 12 wt equivalents of KMnO4 as oxidant, were coded as GO-4, GO-6, GO-8, GO-10 and GO-12, respectively. After incubating sheets with a fixed concentration of BSA at room temperature, interactions were monitored with time. The analysis is based on UV-vis spectroscopy, fluorescence quenching, dynamic light scattering (DLS), small angle neutron scattering (SANS), Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) techniques. Binding of BSA over sheets was recorded in the following order; GO-04 >> GO-06 > GO-08 > GO-10 ≈ GO-12. Our observations suggest that these interactions are largely regulated by the availability of pure graphitic domains and density of oxygen functionalities on sheet surface. This led us to the conclusion that GO-protein interactions can be minimized by modulating the extent of sheet oxidation. Moreover, we show that adsorption of proteins as colloidal aggregates contributes to improved biosafety of sheets. The protein molecule did not exhibit depletive changes in its conformation. However, from the viewpoint of drug delivery applications, density of oxygen groups must be optimized for maximizing the loading efficiency of oxidized sheets.

Fine-tuning miR-21 expression and inhibition of EMT in breast cancer cells using aromatic-neomycin derivatives.

MicroRNAs (miRs) are a class of endogenously expressed non-coding RNAs that negatively regulate gene expression within cells and participate in maintaining cellular homeostasis. By targeting 3' UTRs of target genes, individual miRs can control a wide array of gene expressions. Previous research has shed light upon the fact that aberrantly expressed miRs within cells can pertain to diseased conditions, such as cancer. Malignancies caused due to miRs are because of the high expression of onco-miRs or feeble expression of tumor-suppressing miRs. Studies have also shown miRs to engage in epithelial to mesenchymal transition (EMT), which allows cancer cells to become more invasive and metastasize. miR-21 is an onco-miR highly expressed in breast cancer cells and targets protein PTEN, which abrogates EMT. Therefore, we discuss an approach where in-house-developed peptidic amino sugar molecules have been used to target pre-miR-21 to inhibit miR-21 biogenesis, and hence antagonize its tumor-causing effect and inhibit EMT. Our study shows that small-molecule-based fine-tuning of miR expression can cause genotypic as well as phenotypic changes and also reinstates the potential and importance of nucleic acid therapeutics.

Recent Advances in the Discovery of Antiviral Metabolites from Fungi.

As the world manages the impact of a global pandemic caused by COVID-19, the discovery of new antiviral agents has become way more relevant and urgent. Viruses are submicroscopic infectious agents that replicate inside the living cells of different organisms. These viruses use nucleic acids (both DNA and RNA) for further replication and maturity inside the cells. Some of the viruses responsible for various human and plant diseases belong to the classes of Picornaviridae, Retroviridae, Orthomyxoviridae, Flaviviridae, Pneumoviridae, Virgaviridae, and Hepadnaviridae, and their treatment options are limited or non-existent. The consistent reemergence and resistance development in the viral strains demand the discovery and development of new antiviral drugs possessing better efficacy. Bio-active compounds isolated from fungi can be the source of new compounds with enhanced potency and new mechanisms of action. Fungi are known to produce a diverse lot of secondary metabolites due to their existence in harsh and testing climates which are often inhabitable for many organisms. Because of these unique environments, fungi produce a variety of secondary metabolites of different chemical classes like alkaloids, quinones, furanone, pyrones, benzopyranoids, xanthones, terpenes, steroids, peptides, and many acyclic compounds. Fungal metabolites are known to display a wide range of bioactive attributes, i.e., anticancer, antibacterial, antifungal, and anti-Alzheimer's, along with antiviral properties. In this review article, we report over 300 antiviral compounds from fungal sources during the period of 2009 to 2019. The source of these compounds is marine and endophytic fungi and they are arranged based on their antiviral action against different viral families. These compounds offer promise for their use and development as future antiviral drugs.

Beyond amyloid proteins: Thioflavin T in nucleic acid recognition.

Thioflavin T (ThT) is a commercially available fluorescent dye that is commonly used in biomedical research for over five decades. It was first reported as an extrinsic fluorescent probe for the detection of amyloid fibrils and related processes and it has also been used extensively for assessing protein binding in fluorescence-based assays. Although the nucleic acid binding of ThT was reported half of a century ago in the 1970s, it was not widely explored until the start of this decade. In recent years, Thioflavin T has become a major tool in the recognition of many types of non-canonical nucleic acid conformations including duplexes, triplexes, and G-quadruplexes. The propensity of ThT binding is more towards base aberrations, bulges, and mismatches highlighting its importance in serving as a diagnostic tool in a variety of ailments/disease conditions. In this review, we cover major advancements in nucleic acid detection/binding by ThT to a variety of nucleic acid structures.

Bacterial rRNA A-site recognition by DAPI: Signatures of intercalative binding.

The bacterial A-site RNA is one of the key targets towards the development of new antibacterials including new treatment options for tuberculosis. Using DAPI as a prototype, we have explored the potential of bisamidines as a potential chemical motif for bacterial A-site recognition. We have demonstrated that the binding of DAPI shows a concentration-dependent thermal stabilization of the bacterial A-site RNA (ΔTm = 9.9 °C). The binding, however, does not show pH-dependent changes upon lowering of pH. Both UV-vis and CD experiments show that the DAPI binding involves base stacking with the RNA bases in a manner that is analogous to intercalation. Scatchard analysis of the UV-vis titration data revealed a micromolar affinity of the DAPI to the bacterial rRNA A-Site (Ka = 1.14 × 106 M-1) which was corroborated by the FID-based relative binding affinity comparison with aminoglycosides. The molecular docking studies showed binding poses consistent with polar and stacking interactions with the RNA. These studies highlight the role of amidines in bacterial A-site recognition and the need for the development of their structural analogs towards the making of aminoglycoside mimics.

Selective, pH sensitive, "turn on" fluorescence sensing of carbonate ions by a benzimidazole.

Anions play crucial roles in the sustenance of life on earth in many ways. Selective detection of specific anions is important in developing new diagnostic tools and therapeutics. A pH-sensitive & selective benzimidazole-based fluorescent sensor has been developed for rapid detection of carbonate ions which can detect carbonate ions in low nanomolar concentrations. NMR based experiments indicate direct interaction of benzimidazole imino protons with the carbonate ions leading to 1:1 ligand carbonate ion complexation events. This is one of the first reports of benzimidazole sensing carbonate ions with high selectivity which may have implications in disease prevention and toxicity assessment.

New Approaches in Sensing and Targeting Bacterial rRNA A-site.

New chemical agents that could combat increasing antibiotic resistance are urgently needed. In this mini-review, an old but highly relevant RNA sequence which is crucial for the continuation of bacterial life-cycle is covered. Some of the most significant advances of the last decade in sensing and targeting the bacterial rRNA A-site: a well-validated binding site of proverbially known aminoglycoside antibiotics are described. Some of the major advances in direct sensing of the bacterial decoding side (A-site) are described and also new fluorescent molecules that are capable of detecting lead compounds through high-throughput assays by displacement of fluorescent probe molecules are highlighted. Lastly, some of the recently discovered non-aminoglycoside small molecule binders of bacterial rRNA A-site as a new class of molecules that could provide future scaffolds and molecules for developing new antibacterial agents have been discussed.

Right-sided versus left-sided percutaneous transhepatic biliary drainage in the management of malignant biliary obstruction: a randomized controlled study.

AIM: To compare the technical difficulty, safety, radiation exposure and success rates between right-sided and left-sided percutaneous transhepatic biliary drainage (RPTBD and LPTBD) in patients with malignant biliary obstruction (MBO). MATERIALS AND METHODS: Fifty patients (28 males, 22 females; mean age 51.78 years) with MBO were randomized to undergo either RPTBD or LPTBD during the study period between June 2016 and May 2018. The procedure time, fluoroscopy time, radiation doses to the operators and patients, technical success, clinical success, complications and effect on quality of life were evaluated and compared between the two groups. RESULTS: Twenty-five patients were included in each group. The technical success was 100% in both groups. There was no significant difference between RPTBD and LPTBD groups in terms of major complications [4% and 12%, respectively; p = 0.297] and minor complications [40% and 32%, respectively; p = 0.597]. Further, the average procedure time (37.80 ± 13.07 min vs 41.04 ± 14.94 min), fluoroscopy time (5.88 ± 4.2 min vs 5.97 ± 3.8 min), radiation doses to the operator (136.84 ± 106.67 µSv vs 130.40 ± 106.46 µSv) and to the patient (8.23 ± 5.80 Gycm2 vs 11.74 ± 11.28 Gycm2) were not significantly different between the groups. Clinical success was achieved in 21 patients (84%) of RPTBD group and 17 patients (68%) of LPTBD group with no significant difference (p = 0.416) between them. CONCLUSION: There was no significant difference between RPTBD and LPTBD with reference to the technique, safety, radiation dose, success rates and impact on quality of life suggesting no laterality advantage for biliary drainage in cases of MBO.

Surface Dependent Dual Recognition of a G-quadruplex DNA With Neomycin-Intercalator Conjugates.

G-quadruplexes have been characterized as structures of vital importance in the cellular functioning of several life forms. They have subsequently been established to serve as a therapeutic target of several diseases including cancer, HIV, tuberculosis and malaria. In this paper, we report the binding of aminosugar-intercalator conjugates with a well-studied anti-parallel G-quadruplex derived from Oxytricha Nova G-quadruplex DNA. Of the four neomycin-intercalator conjugates studied with varying surface areas, BQQ-neomycin conjugate displayed the best binding to this DNA G-quadruplex structure with an association constant of K a = (1.01 ±0.03) × 107 M-1 which is nearly 100-fold higher than the binding of neomycin to this quadruplex. The binding of BQQ-neomycin displays a binding stoichiometry of 1:1 indicating the presence of a single and unique binding site for this G-quadruplex. In contrast, the BQQ-neomycin displays very weak binding to the bacterial A-site rRNA sequence showing that BQQ-does not enhance the neomycin binding to its natural target, the bacterial rRNA A-site. The BQQ-neomycin conjugate is prone to aggregation even at low micromolar concentrations (4 µM) leading to some ambiguities in the analysis of thermal denaturation profiles. Circular dichroism experiments showed that binding of BQQ-neomycin conjugate causes some structural changes in the quadruplex while still maintaining the overall anti-parallel structure. Finally, the molecular docking experiments suggest that molecular surface plays an important role in the recognition of a second site on the G-quadruplex. Overall, these results show that molecules with more than one binding moieties can be made to specifically recognize G-quadruplexes with high affinities. The dual binding molecules comprise of quadruplex groove binding and intercalator units, and the molecular surface of the intercalator plays an important part in enhancing binding interaction to the G-quadruplex structure.

Recent Advances in Therapeutic Applications of Bisbenzimidazoles.

Nitrogen-containing heterocycles are one of the most common structural motifs in approximately 80% of the marketed drugs. Of these, benzimidazoles analogues are known to elicit a wide spectrum of pharmaceutical activities such as anticancer, antibacterial, antiparasitic, antiviral, antifungal as well as chemosensor effect. Based on the benzimidazole core fused heterocyclic compounds, crescent-shaped bisbenzimidazoles were developed which provided an early breakthrough in the sequence-specific DNA recognition. Over the years, a number of functional variations in the bisbenzimidazole core have led to the emergence of their unique properties and established them as versatile ligands against several classes of pathogens. The present review provides an overview of diverse pharmacological activities of the bisbenzimidazole analogues in the past decade with a brief account of its development through the years.

Gram-negative synergy and mechanism of action of alkynyl bisbenzimidazoles.

Bisbenzimidazoles with terminal alkynyl linkers, selective inhibitors of bacterial topoisomerase I, have been evaluated using bacterial cytological profiling (BCP) to ascertain their mechanism of action and screened for synergism to improve Gram-negative bacterial coverage. Principal component analysis of high throughput fluorescence images suggests a dual-mechanism of action affecting DNA synthesis and cell membrane integrity. Fluorescence microscopy of bacteria challenged with two of the alkynyl-benzimidazoles revealed changes in the cellular ultrastructure that differed from topoisomerase II inhibitors including induction of spheroplasts and membrane lysis. The cytoskeleton recruitment enzyme inhibitor A22 in combination with one of the alkynyl-benzimidazoles was synergistic against Acinetobacter baumannii and Escherichia coli. Gram-positive coverage remained unchanged in the A22-alkynyl bisbenzimidazole combination. Efflux inhibitors were not synergistic, suggesting that the Gram-negative outer membrane was a significant barrier for alkynyl-bisbenzimidazole uptake. Time-kill assays demonstrated the A22-bisbenzimidazole combination had a similar growth inhibition curve to that of norfloxacin in E.coli. Bisbenzimidazoles with terminal alkynyl linkers likely impede bacterial growth by compromising cell membrane integrity and by interfering with DNA synthesis against Gram-positive pathogens and in the synergistic combination against Gram-negative pathogens including E. coli and multidrug-resistant A. baumanii.

A fluorescent aminosugar to rapidly screen and study RNA binders.

RNA targeted high-throughput assays that allow for rapid detection of high affinity binding ligands are important in RNA recognition studies. A number for fluorescent dyes have been reported that can assist in rapidly identifying nucleic acid (RNA) binding elements without the need for immobilization of RNA or the ligand. A number of these dyes are planar aromatic molecules that bind non-specifically to nucleic acids and often distort their parent nucleic acid structures leading to ambiguity in the interpretation of results. In this light, we report here, the use of an aminoglycoside (neomycin) based fluorescent probe (F-Neo) which can reversibly bind to different RNA motifs and help identify ligands with needed affinity and selectivity, without any immobilization of the probe or the target. In this chapter, we provide the details of the assay development, experimental considerations and data analysis to use the probe and identify novel ligands. We then provide a brief introduction to calorimetry (ITC) and circular dichroism (CD) spectroscopy based methods in validating the binding of such identified compounds.