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.

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.

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.

Spectroscopic studies of Thioflavin-T binding to c-Myc G-quadruplex DNA.

G-quadruplexes are well-known DNA secondary structures which can be formed both within the DNA and the RNA sequences of the human genome. While many functions of G-quadruplex during cell regulatory events are still unknown, a number of reports have established their role in finding new cancer therapies. In this report, we provide a detailed account of Thioflavin T (ThT) interacting with a promoter gene (c-Myc) which has relevance in several types of human cancers. Using a variety of spectroscopic techniques, we have shown that the binding of ThT is selective to c-Myc G-quadruplex only, having poor interactions with the duplex DNA sequences. UV-Visible titration experiments show that binding involves stacking interactions which were further corroborated by CD experiments. Fluorescence studies showed that the binding of ThT to c-Myc G-quadruplex results in a large increase in the fluorescence emission spectrum of c-Myc G-quadruplex while the same to duplex DNAs was much poor. Binding of ThT to c-Myc G-quadruplex results in thermal stabilization of the quadruplex DNA by up to 7.4 °C and Job plot experiments demonstrated the presence of 1:1 and 2:1 ligand to quadruplex complexes. Finally, the docking study suggested that ThT stacks with the guanine bases in one of the grooves which is in agreement with the CD studies. These results are expected to provide leads into the design of new ThT analogs and derivatives for enhancing the stability and selectivity of new G-quadruplex targeting ligands.

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.

Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase I by Hoechst 33258 Derived Mono- and Bisbenzimidazoles.

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC50 values <5.0 µM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 µg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 µg/mL, Klebsiella pneumoniae > 32 µg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2-23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.

Marine Fungi: A Source of Potential Anticancer Compounds.

Metabolites from marine fungi have hogged the limelight in drug discovery because of their promise as therapeutic agents. A number of metabolites related to marine fungi have been discovered from various sources which are known to possess a range of activities as antibacterial, antiviral and anticancer agents. Although, over a thousand marine fungi based metabolites have already been reported, none of them have reached the market yet which could partly be related to non-comprehensive screening approaches and lack of sustained lead optimization. The origin of these marine fungal metabolites is varied as their habitats have been reported from various sources such as sponge, algae, mangrove derived fungi, and fungi from bottom sediments. The importance of these natural compounds is based on their cytotoxicity and related activities that emanate from the diversity in their chemical structures and functional groups present on them. This review covers the majority of anticancer compounds isolated from marine fungi during 2012-2016 against specific cancer cell lines.

Potent inhibition of miR-27a by neomycin-bisbenzimidazole conjugates.

miRNAs are important components of regulatory networks that control gene expression and have implications in various diseases including cancer. Targeting oncogenic miRNAs with small molecules is currently being explored to develop cancer therapeutics. Here, we report the development of dual binding neomycin-bisbenzimidazole conjugates that target oncogenic miR-27a with high affinity (Ka = 1.2 to 7.4 × 108 M-1). These conjugates bring significant reduction (∼65% at 5 µM) in mature miRNA levels and penetrate easily in the cells where they localise both in the cytoplasm and the nucleus. Cell cycle analysis showed significant increase in the G0/G1 phase (∼15%) and decrease in the S phase (∼7%) upon treatment with neomycin-bisbenzimidazole conjugates, suggesting inhibition of cell proliferation. Using the conjugation approach, we show that moderately binding ligands can be covalently combined into high affinity binders. This study also highlights the role of linker optimization in designing high affinity ligands for miR-27a targeting.

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.

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(+).

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.

Aminoglycoside binding to Oxytricha nova telomeric DNA.

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ∼ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.

Evaluation of the stability of extemporaneously prepared ophthalmic formulation of mitomycin C.

Mitomycin C (MMC) is a cytostatic agent topically used in conjunctival neoplasms, secondary to glaucoma filtering, pterygium, and strabismus surgery to increase the success rate. The topical formulation of MMC for ocular use is always extemporaneously prepared. Our study evaluated the stability of extemporaneously prepared formulations of MMC at different concentrations (150, 300, & 600 microg/mL) kept at different temperatures (25 degrees , 4 degrees , and -70 degrees C) and at different pH range (6, 7, and 8). Aliquots from the above formulations were subjected for quantification of MMC on days 0, 7, 14, 21, and 28 using high-performance liquid chromatography. MMC stored at 25 degrees C for 6 months was also subjected to flow cytometry and compared to freshly prepared MMC. The results indicated that the degradation of MMC is very high in acidic pH at room temperature. Increasing the pH to 7 or 8 and keeping MMC at low temperatures significantly decreased the degradation of MMC. Interestingly, the flow cytometry data revealed that the 6-month-old MMC showed an antiproliferative effect compared to that of freshly prepared MMC. To conclude, the extemporaneously prepared MMC at pH between 7 and 8 and stored in the refrigerator can increase the duration of its stability. However, the antiproliferative study using flow cytometry revealed that degraded MMC retained its activity even after degradation.