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.

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.

An overview of recent advances in duplex DNA recognition by small molecules.

As the carrier of genetic information, the DNA double helix interacts with many natural ligands during the cell cycle, and is amenable to such intervention in diseases such as cancer biogenesis. Proteins bind DNA in a site-specific manner, not only distinguishing between the geometry of the major and minor grooves, but also by making close contacts with individual bases within the local helix architecture. Over the last four decades, much research has been reported on the development of small non-natural ligands as therapeutics to either block, or in some cases, mimic a DNA-protein interaction of interest. This review presents the latest findings in the pursuit of novel synthetic DNA binders. This article provides recent coverage of major strategies (such as groove recognition, intercalation and cross-linking) adopted in the duplex DNA recognition by small molecules, with an emphasis on major works of the past few years.

Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment.

Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common platform fragment-based drug discovery, two fragments that bind to adjacent sites on a target are identified and are then linked together using different linkers to identify the linkage for optimum activity. What are not known from these studies are the effects these linkers, which typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, and photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258-modeled bisbenzimidazoles (1-9) that contain linkers of varying lengths (3-21 atoms) display length- and composition-dependent variation in B-DNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3 to 9.0 °C as the linker length increased from 3 to 21 atoms, respectively. Compounds with linker lengths of ≤11 atoms (such as compounds 1 and 5) are localized in the nucleus, while compounds with long linkers (such as compounds 8 and 9) are distributed in the extranuclear space, as well, with possible interactions with extranuclear targets. These findings provide insights into future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.

Linker dependent intercalation of bisbenzimidazole-aminosugars in an RNA duplex; selectivity in RNA vs. DNA binding.

Neomycin and Hoechst 33258 are two well-known nucleic acid binders that interact with RNA and DNA duplexes with high affinities respectively. In this manuscript, we report that covalent attachment of bisbenzimidazole unit derived from Hoechst 33258 to neomycin leads to intercalative binding of the bisbenzimidazole unit (oriented at 64-74° with respected to the RNA helical axis) in a linker length dependent manner. The dual binding and intercalation of conjugates were supported by thermal denaturation, CD, LD and UV-Vis absorption experiments. These studies highlight the importance of linker length in dual recognition by conjugates, for effective RNA recognition, which can lead to novel ways of recognizing RNA structures. Additionally, the ligand library screens also identify DNA and RNA selective compounds, with compound 9, containing a long linker, showing a 20.3°C change in RNA duplex Tm with only a 13.0°C change in Tm for the corresponding DNA duplex. Significantly, the shorter linker in compound 3 shows almost the reverse trend, a 23.8°C change in DNA Tm, with only a 9.1°C change in Tm for the corresponding RNA duplex.

Multivalency in the recognition and antagonism of a HIV TAR RNA-TAT assembly using an aminoglycoside benzimidazole scaffold.

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA with a low nanomolar affinity. 5 displayed enhanced binding compared to its individual building blocks including the neomycin dimer azide and benzimidazole alkyne. In essence, a high affinity multivalent ligand was designed and synthesized to target HIV TAR RNA.

Analysis of diazofluorene DNA binding and damaging activity: DNA cleavage by a synthetic monomeric diazofluorene.

The lomaiviticins and kinamycins are complex DNA damaging natural products that contain a diazofluorene functional group. Herein, we elucidate the influence of skeleton structure, ring and chain isomerization, D-ring oxidation state, and naphthoquinone substitution on DNA binding and damaging activity. We show that the electrophilicity of the diazofluorene appears to be a significant determinant of DNA damaging activity. These studies identify the monomeric diazofluorene 11 as a potent DNA cleavage agent in tissue culture. The simpler structure of 11 relative to the natural products establishes it as a useful lead for translational studies.

Enhanced Sequence-Specific DNA Recognition Using Oligodeoxynucleotide-Benzimidazole Conjugates.

Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties. In the present study, five Hoechst 33258 derived benzimidazoles were conjugated to T rich ODNs and their hybridization effectiveness was tested. Thermal denaturation studies revealed significant stabilization of complementary duplexes by ODN-benzimidazole conjugates, with the extent of stabilization being highly dependent on the length of the linker between DNA and benzimidazole. The increases in thermal stability were determined to be due to the binding of the benzimidazole moiety to the duplex. Circular dichroism and molecular modeling studies provided insights toward the influence of conjugation on duplex structure and how linker length impacts placement of the benzimidazole moiety in the minor groove. Furthermore, thermal denaturation studies with the complementary strand containing a single base mismatch or being RNA revealed that covalent conjugation of benzimidazoles to an ODN also enhances the sequence specificity. The fundamental studies reported herein provide a strategy to improve the stability and specificity properties of the ODN probes, which can be of use for targeting and diagnostics applications.

Characterization of ribosomal binding and antibacterial activities using two orthogonal high-throughput screens.

We report here the affinity and antibacterial activity of a structurally similar class of neomycin dimers. The affinity of the dimer library for rRNA was established by using a screen that measures the displacement of fluorescein-neomycin (F-neo) probe from RNA. A rapid growth inhibition assay using a single drug concentration was used to examine the antibacterial activity. The structure-activity relationship data were then rapidly analyzed using a two-dimensional ribosomal binding-bacterial inhibition plot analysis.

An assay for human telomeric G-quadruplex DNA binding drugs.

Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K(+) or Na(+).

Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

Targeting C-myc G-quadruplex: dual recognition by aminosugar-bisbenzimidazoles with varying linker lengths.

G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

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.

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.

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.

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.

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.

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.

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.

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.