ABSTRACT
Recognition of nucleic acids remains an important endeavor in biology. Nucleic acids adopt shapes ranging from A-form (RNA and GC rich DNA) to B-form (AT rich DNA). We show, in this contribution, shape-specific recognition of A-U rich RNA duplex by a neomycin (Neo)-polydiacetylene (PDA) complex. PDA assemblies are fabricated by using a well-known diacetylene (DA) monomer, 10,12-pentacosadiynoic acid (PCDA). The response of poly(PCDA) assemblies is generated by mixing with a modified neomycin-PCDA monomer (Neo-PCDA). The functionalization by neomycin moiety provides specific binding with homopolyribonucleotide poly (rA) - poly (rU) stimulus. Various types of alcohols are utilized as additives to enhance the sensitivity of poly(PCDA)/Neo-PCDA assemblies. A change of absorption spectra is clearly observed when a relatively low concentration of poly (rA)-poly (rU) is added into the system. Furthermore, poly(PCDA)/Neo-PCDA shows a clear specificity for poly (rA)-poly (rU) over the corresponding DNA duplex. The variation of linker between neomycin moiety and conjugated PDA backbone is found to significantly affect its sensitivity. We also investigate other parameters including the concentration of Neo-PCDA and the DA monomer structure. Our results provide here preliminary data for an alternative approach to improve the sensitivity of PDA utilized in biosensing and diagnostic applications.
ABSTRACT
Nucleic acids adopt a broad array of hydrogen-bonded structures that enable their diverse roles in the cell; even the familiar DNA double helix displays subtle architectural nuances that are sequence dependent. While there have been many approaches for recognition of B-form nucleic acids, A-form DNA recognition has lagged behind. Here, using a tight binding fluorescein-neomycin (F-neo) conjugate that can probe the electrostatic environment of A-form DNA major groove, we developed a fluorescent displacement assay to be used as a screen for DNA duplex-binding compounds. As opposed to intercalating dyes that can significantly perturb DNA structure, the groove binding F-neo allows the probing of native DNA conformation. In combination with the assay development and probing of DNA grooves, we also report the synthesis and binding of a series of neomycin-anthraquinone conjugates, two units with a known preference for binding GC rich DNA. The assay can be used to identify duplex DNA-binding compounds, as well as probe structural features of a target DNA duplex, and can easily be scaled up for high throughput screening of compound libraries.
Subject(s)
DNA, A-Form/analysis , Fluorescein/chemistry , Fluorescent Dyes/analysis , Neomycin/chemistry , Molecular Docking Simulation , Molecular StructureABSTRACT
The antibacterial effects of aminoglycosides are based on their association with the A-site of bacterial rRNA and interference with the translational process in the bacterial cell, causing cell death. The clinical use of aminoglycosides is complicated by resistance and side effects, some of which arise from their interactions with the human mitochondrial 12S rRNA and its deafness-associated mutations, C1494U and A1555G. We report a rapid assay that allows screening of aminoglycoside compounds to these classes of rRNAs. These screening tools are important to find antibiotics that selectively bind to the bacterial A-site rather than human, mitochondrial A-sites and its mutant homologues. Herein, we report our preliminary work on the optimization of this screen using 12 anthraquinone-neomycin (AMA-NEO) conjugates against molecular constructs representing five A-site homologues, Escherichia coli, human cytosolic, mitochondrial, C1494U, and A1555G, using a fluorescent displacement screening assay. These conjugates were also tested for inhibition of protein synthesis, antibacterial activity against 14 clinically relevant bacterial strains, and the effect on enzymes that inactivate aminoglycosides. The AMA-NEO conjugates demonstrated significantly improved resistance against aminoglycoside-modifying enzymes (AMEs), as compared with NEO. Several compounds exhibited significantly greater inhibition of prokaryotic protein synthesis as compared to NEO and were extremely poor inhibitors of eukaryotic translation. There was significant variation in antibacterial activity and MIC of selected compounds between bacterial strains, with Escherichia coli, Enteroccocus faecalis, Citrobacter freundii, Shigella flexneri, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Staphylococcus epidermidis, and Listeria monocytogenes exhibiting moderate to high sensitivity (50-100% growth inhibition) whereas Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellla pneumoniae, and MRSA strains expressed low sensitivity, as compared to the parent aminoglycoside NEO.
Subject(s)
Aminoglycosides/pharmacology , Anti-Infective Agents/pharmacology , RNA, Ribosomal/antagonists & inhibitors , Aminoglycosides/chemistry , Anthraquinones/chemistry , Anthraquinones/pharmacology , Anti-Infective Agents/chemistry , Binding Sites , Drug Resistance, Microbial/drug effects , Humans , Microbial Sensitivity Tests , Mutation , Neomycin/chemistry , Neomycin/pharmacology , RNA, Bacterial/antagonists & inhibitors , RNA, Bacterial/chemistry , RNA, Ribosomal/chemistry , RNA, Ribosomal/geneticsABSTRACT
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.
Subject(s)
Aminoglycosides/pharmacology , Benzimidazoles/pharmacology , HIV Long Terminal Repeat/drug effects , RNA, Viral/antagonists & inhibitors , Aminoglycosides/chemical synthesis , Aminoglycosides/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Binding Sites/drug effects , Click Chemistry , Dose-Response Relationship, Drug , Ligands , Molecular Conformation , Neomycin/chemistry , Neomycin/pharmacology , Structure-Activity RelationshipABSTRACT
A 215-member mono- and diamino acid peptidic-aminosugar (PA) library, with neomycin as the model aminosugar, was systematically and rapidly synthesized via solid phase synthesis. Antibacterial activities of the PA library, on 13 bacterial strains (seven Gram-positive and six Gram-negative bacterial strains), and binding affinities of the PA library for a 27-base model of the bacterial 16S ribosomal A-site RNA were evaluated using high-throughput screening. The results of the two assays were correlated using Ribosomal Binding-Bacterial Inhibition Plot (RB-BIP) analysis to provide structure-activity relationship (SAR) information. From this work, we have identified PAs that can discriminate the E. coli A-site from the human A-site by up to a 28-fold difference in binding affinity. Aminoglycoside-modifying enzyme activity studies indicate that APH(2â³)-Ia showed nearly complete removal of activity with a number of PAs. The synthesis of the compound library and screening can both be performed rapidly, allowing for an iterative process of aminoglycoside synthesis and screening of PA libraries for optimal binding and antibacterial activity for lead identification.
Subject(s)
Amino Sugars/chemistry , Anti-Bacterial Agents/pharmacology , Peptide Library , RNA/metabolism , Anti-Bacterial Agents/metabolism , Bacteria/drug effects , Bacteria/growth & development , Carbohydrate Sequence , High-Throughput Screening Assays , Microbial Sensitivity Tests , Molecular Sequence Data , Structure-Activity RelationshipABSTRACT
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(+).
Subject(s)
Biological Assay/methods , G-Quadruplexes , Benzothiazoles/chemistry , Benzothiazoles/metabolism , Humans , Kinetics , Ligands , Neomycin/chemistry , Neomycin/metabolism , Quinolines/chemistry , Quinolines/metabolism , Spectrometry, Fluorescence , Telomere/metabolismABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Ribosomes/chemistry , Enterobacter cloacae/drug effects , Microbial Sensitivity Tests , Molecular Structure , Neomycin/chemistry , Neomycin/pharmacology , Pseudomonas aeruginosa/drug effects , Serratia marcescens/drug effects , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
Four novel porphyrins containing nitric oxide (NO) donors were synthesized, and the structures of all the products were characterized by IR, UV-vis, (1)H NMR, and elementary analysis. Interestingly, these new compounds not only were able to release NO, but also showed cancer cell-oriented accumulation. Higher accumulation of these new porphyrins containing NO donors in BEL-7402 liver cancer cells than in L-02 liver normal cells was corroborated by UV-vis spectroscopy. The biological activity of these porphyrins against BEL-7402 liver cancer cells was tested with a MTT assay. The studies indicated that they had more effective killing of BEL-7402 liver cancer cells than that of L-02 liver normal cells, and they had similar activity against MCF-7 breast cancer cells when compared to 5-fluorouracil in the absence of light.