Thermodynamics of d(GGGGCCCC) Binding to Neomycin-Class Aminoglycosides.

DNA adopts a number of conformations that can affect its binding to other macromolecules. The conformations (A, B, Z) can be sequence- and/or solution-dependent. While AT-rich DNA sequences generally adopt a Canonical B-form structure, GC-rich sequences are more promiscuous. Recognition of GC-rich nucleic acids by small molecules has been much more challenging than the recognition of AT-rich duplexes. Spectrophotometric and calorimetric techniques were used to characterize the binding of neomycin-class aminoglycosides to a GC-rich DNA duplex, G4C4, in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of G4C4, with thermal enhancement decreasing with increasing pH and/or Na+ concentration. Although G4C4 bound to aminoglycosides demonstrated a mixed A- and B-form conformation, circular dichroism studies indicate that binding induces a conformational shift toward A-form DNA. Isothermal titration calorimetry studies reveal that aminoglycoside binding to G4C4 is linked to the uptake of protons at pH = 7.0 and that this uptake is pH-dependent. Increased pH and/or Na+ concentration results in a decrease in G4C4 affinity for the aminoglycosides. The binding affinities of the aminoglycosides follow the expected hierarchy: neomycin > paromomycin > ribostamycin. The salt dependence of DNA binding affinities of aminoglycosides is consistent with at least two drug NH3+ groups participating in electrostatic interactions with G4C4. These studies further embellish our understanding of the many factors facilitating recognition of GC-rich DNA structures as guided by their optimum charge and shape complementarity for small-molecule amino sugars.

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19 F NMR.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states.

RNA Chemical Labeling with Site-Specific, Relative Quantification by Mass Spectrometry for the Structural Study of a Neomycin-Sensing Riboswitch Aptamer Domain.

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.

Design, Synthesis, and Evaluation of Neomycin-Imidazole Conjugates for RNA Cleavage.

Targeting RNA with synthetic small molecules attracted much interest during recent years as a particularly promising therapeutic approach in a large number of pathologies spanning from genetic disorders, cancers as well as bacterial and viral infections. In this work, we took advantage of a known RNA binder, neomycin, to prepare neomycin-imidazole conjugates mimicking the active site of ribonuclease enzymes able to induce a site-specific cleavage of HIV-1 TAR RNA in physiological conditions. These new conjugates were prepared using a straightforward synthetic methodology and were studied for their ability to bind the target, inhibit Tat/TAR interaction and induce selective cleavage using fluorescence-based assays and molecular docking. We found compounds with nanomolar affinity, promising cleavage activity and the ability to inhibit Tat/TAR interaction with submicromolar IC50 s.

Targeted modifications of neomycin and paromomycin: Towards resistance-free antibiotics?

Despite their clinical importance, saving numerous human lifes, over- and mis-uses of antibiotics have created a strong selective pressure on bacteria, which induces the emergence of (multi)resistant strains. Antibioresistance is becoming so pregnant that since 2017, WHO lists bacteria threatening most human health (AWaRe, ESKAPE lists), and those for which new antibiotics are urgently needed. Since the century turn, this context is leading to a burst in the chemical synthesis of new antibiotics, mostly derived from natural antibiotics. Among them, aminoglycosides, and especially the neomycin family, exhibit broad spectrum of activity and remain clinically useful drugs. Therefore, numerous endeavours have been undertaken to modify aminoglycosides with the aim of overcoming bacterial resistances. After having replaced antibiotic discovery into an historical perspective, briefly surveyed the aminoglycoside mode of action and the associated resistance mechanisms, this review emphasized the chemical syntheses performed on the neomycin family and the corresponding structure activity relationships in order to reveal the really efficient modifications able to convert neomycin and its analogues into future drugs. This review would help researchers to strategically design novel aminoglycoside derivatives for the development of clinically viable drug candidates.

Design and characterization of 3D printed, neomycin-eluting poly-L-lactide mats for wound-healing applications.

This study evaluates the suitability of 3D printed biodegradable mats to load and deliver the topical antibiotic, neomycin, for up to 3 weeks in vitro. A 3D printer equipped with a hot melt extruder was used to print bandage-like wound coverings with porous sizes appropriate for cellular attachment and viability. The semicrystalline polyester, poly-l-lactic acid (PLLA) was used as the base polymer, coated (post-printing) with polyethylene glycols (PEGs) of MWs 400 Da, 6 kDa, or 20 kDa to enable manipulation of physicochemical and biological properties to suit intended applications. The mats were further loaded with a topical antibiotic (neomycin sulfate), and cumulative drug-release monitored for 3 weeks in vitro. Microscopic imaging as well as Scanning Electron Microscopy (SEM) studies showed pore dimensions of 100 × 400 µm. These pore dimensions were achieved without compromising mechanical strength; because of the "tough" individual fibers constituting the mat (Young's Moduli of 50 ± 20 MPa and Elastic Elongation of 10 ± 5%). The in vitro dissolution study showed first-order release kinetics for neomycin during the first 20 h, followed by diffusion-controlled (Fickian) release for the remaining duration of the study. The release of neomycin suggested that the ability to load neomycin on to PLLA mats increases threefold, as the MW of the applied PEG coating is lowered from 20 kDa to 400 Da. Overall, this study demonstrates a successful approach to using a 3D printer to prepare porous degradable mats for antibiotic delivery with potential applications to dermal regeneration and tissue engineering. Illustration of the process used to create and characterize 3D printed PLLA mats.

Multifunctional Neomycin-Triazine-Based Cationic Lipids for Gene Delivery with Antibacterial Properties.

Cationic lipids (CLs) have gained significant attention among nonviral gene delivery vectors due to their ease of synthesis and functionalization with multivalent moieties. In particular, there is an increasing request for multifunctional CLs having gene delivery capacity and antibacterial activity. Herein, we describe the design and synthesis of a novel class of aminoglycoside (AG)-based multifunctional vectors with high transfection efficiency and noticeable antibacterial properties. Specifically, cationic amphiphiles were built on a triazine scaffold, allowing for an easy derivatization with up to three potentially different substituents, such as neomycin (Neo) that serves as the polar head and one or two lipophilic tails, namely stearyl (ST) and oleyl (OL) alkyl chains and cholesteryl (Chol) tail. With the aim to shed more light on the effect of different types and numbers of lipophilic moieties on the ability of CLs to condense and transfect cells, the performance of Neo-triazine-based derivatives as gene delivery vectors was evaluated and compared. The ability of Neo-triazine-based derivatives to act as antimicrobial agents was evaluated as well. Neo-triazine-based CLs invariably exhibited excellent DNA condensation ability, even at a low charge ratio (CR, +/-). Besides, each derivative showed very good transfection performance at its optimal CR on two different cell lines, along with negligible cytotoxicity. CLs bearing symmetric two-tailed OL proved to be the most effective in transfection. Interestingly, Neo-triazine-based derivatives, used as either free lipids or lipoplexes, exhibited strong antibacterial activity against Gram-negative bacteria, especially in the case of CLs bearing one or two aliphatic chains. Altogether, these results highlight the potential of Neo-triazine-based derivatives as effective multifunctional nonviral gene delivery vectors.

Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches.

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5' untranslated region of mRNA. Rearrangements of the RNA structure in response, e.g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modelling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behaviour in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyse the influence of 5' hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free licence on GitHub (https://github.com/xileF1337/riboswitch_design).

Component Optimization of Neomycin Biosynthesis via the Reconstitution of a Combinatorial Mini-Gene-Cluster in Streptomyces fradiae.

Neomycin, a multicomponent aminoglycoside antibiotic, is mainly utilized in livestock husbandry and feed additives in animals. The antimicrobial potency of the main product neomycin B is higher than that of its stereoisomer neomycin C. However, the content of neomycin C as an impurity in the high-producing strain is relatively high, and its isolation or removal from neomycin B is quite difficult, which influences the widespread application of neomycin. In this work, the essential genes responsible for neomycin biosynthesis were evaluated and overexpressed to reduce the content of neomycin C. Among them, neoG and neoH are two novel regulatory genes for neomycin biosynthesis, aphA is a resistance gene, neoN encoding a radical SAM-dependent epimerase is responsible for the conversion of neomycin C to B using SAM as the cofactor, and metK is a SAM synthetase coding gene. We demonstrated that the reconstitution and overexpression of a mini-gene-cluster (PkasO*-neoN-metK-PkasO*-neoGH-aphA) could effectively reduce the accumulation of neomycin C from 19.1 to 12.7% and simultaneously increase neomycin B by ∼13.1% in the engineered strain Sf/pKCZ04 compared with the wild-type strain (Sf). Real-time quantitative polymerase chain reaction analysis revealed the remarkable up-regulation of the neoE, neoH, neoN, and metK genes situated in the mini-gene-cluster. The findings will pave a new path for component optimization and the large-scale industrial production of significant commercial antibiotics.

Integrated nanotechnology of synergism-sterilization and removing-residues for neomycin through nano-Cu2O.

The abuse of antibiotics has led to widespread antimicrobial resistance (AMR) and environmental pollution. In order to solve these problems, a lot of studies have been carried out mainly focusing on the modification and recombination of organic reagents, but bacteria are still easy to adapt to it, so they cannot be thoroughly solved. Here, we present an integrated pollution-free synergistic antibacterial nanotechnology using inorganic nano-Cu2O, which could not only enhance the efficacy of aminoglycoside antibiotics, but also eliminate their environmental pollution by photocatalytic degradation. It was found that Cu2O showed significantly synergistic antibacterial effect (1+1>2) when combined with aminoglycoside antibiotics against Escherichia coli. The inhibition zone area increased by 59.0% when Cu2O combined with neomycin. This reduces dosage and the risk of AMR, and does not pollute the environment after degradation. Next, to explore the synergistic mechanisms, we have studied the interaction of antibiotics with nanoparticles, as well as the interaction of antibacterial agents with bacteria. At last, we believe that the destruction of cell walls by Cu2O facilitates the entry of antibiotics into cells is the reason for their synergy.

Modification at the 2'-Position of the 4,5-Series of 2-Deoxystreptamine Aminoglycoside Antibiotics To Resist Aminoglycoside Modifying Enzymes and Increase Ribosomal Target Selectivity.

A series of derivatives of the 4,5-disubstituted class of 2-deoxystreptamine aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin was prepared and assayed for (i) their ability to inhibit protein synthesis by bacterial ribosomes and by engineered bacterial ribosomes carrying eukaryotic decoding A sites, (ii) antibacterial activity against wild type Gram negative and positive pathogens, and (iii) overcoming resistance due to the presence of aminoacyl transferases acting at the 2'-position. The presence of five suitably positioned residual basic amino groups was found to be necessary for activity to be retained upon removal or alkylation of the 2'-position amine. As alkylation of the 2'-amino group overcomes the action of resistance determinants acting at that position and in addition results in increased selectivity for the prokaryotic over eukaryotic ribosomes, it constitutes an attractive modification for introduction into next generation aminoglycosides. In the neomycin series, the installation of small (formamide) or basic (glycinamide) amido groups on the 2'-amino group is tolerated.

Synthesis and characterization of polyaniline-drug conjugates as effective antituberculosis agents.

Polyaniline (PANI) and its drug composites with some drugs like Neomycin (NM), Trimethoprim (TMP) and Streptomycin (ST) have been prepared by oxidative polymerization of aniline using hydrochloric acid (HA) and ammonium persulfate (APS) as a dopant and as an oxidant, respectively. The structures of PANI and PANI-drug composites were elucidated by FTIR and NMR spectroscopy, which confirmed the presence of benzenoid and quinoid rings in the synthesized compound. Molecular weight and thermal stability were determined by gel permeation chromatography (GPC) and thermogarvimetric analysis, respectively. From the GPC, PDI values of PANI-NM, PANI-TMP and PANI-ST were found to be 1.37, 1.23 and 1.56, respectively. For the study of antibacterial behavior of the synthesized PANI and PANI-drug composites, different micro-organisms, namely, four Gram positive (S. aureus MTCC 96, B. subtilis MTCC 441, S. pyogenes MTCC 442 and S. mutans MTCC 890) and four Gram negative (S. typhi MTCC 98, KL. pneumoniae MTCC 109, E. coli MTCC 443 and P. aeruginosa MTCC 1688) bacteria were selected due to their pharmacological importance. Some of the PANI-drug composites were found to show excellent results as compared to components polyaniline and drugs used for composite formation. Antituberculosis activity of the PANI and its drug composites against Mycobacterium tuberculosisH37RV (acid fast Bacilli) was determined. MIC values for PANI-NM and PANI-TMP were found to be 0.12 and 0.20 µg/mL, respectively. Results suggested that some of the drug composites may be tried as potential candidates for use as an antituberculoid agent to reduce TB transmission.

Aminoglycoside Functionalization as a Tool for Targeting Nucleic Acids.

Aminoglycoside functionalization as a tool for targeting natural and unnatural nucleic acids holds great promise in their development as diagnostic probes and medicinally relevant compounds. Simple synthetic procedures designed to easily and quickly manipulate amino sugar (neomycin, kanamycin) to more powerful and selective ligands are presented in this chapter. We describe representative procedures for (a) aminoglycoside conjugation and (b) preliminary screening for their nucleic acid binding and selectivity.

Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

Enzyme-oxidative-polymerization method for improving glycosaminoglycans stability and reducing calcification in bioprosthetic heart valves.

Glutaraldehyde (GLUT) crosslinked bioprosthetic heart valves (BHVs) might fail within ten years due to progressive degradation and calcification. GLUT cannot stabilize glycosaminoglycans (GAGs), one important component of BHVs. In this current study we developed a new BHVs preparation strategy named as 'HPA/NT/HRP' treatment that utilized 3,4-hydroxyphenylpropionic acid (HPA)/tyramine (TRA)/neomycin trisulfate (NT) conjugated pericardiums and horseradish peroxidase (HRP)/H2O2 enzyme-oxidative-polymerization method. HPA/TRA-pericardium and HPA-NT conjugation would provide extra phenol groups for enzymatic crosslinking. HPA/TRA conjugated pericardium could be crosslinked by HRP/H2O2 enzyme-oxidative-polymerization. The feeding ratio of HPA-NT was optimized. The GAGs content, collagenase and elastase degradation in vitro, the in vivo GAGs stability and anti-calcification potential of HPA/NT/HRP treated pericardiums were characterized. We demonstrated that HPA/NT/HRP treated pericardiums had sufficiently increased GAGs stabilization and decreased calcification. This new HPA/NT/HRP enzyme-oxidative-polymerization strategy would be a promising method to make BHVs with better GAGs stability and anti-calcification properties.

Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer.

The ability of the cytidine analog Çmf to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Çmf-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Çmf. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Çmf at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.

A fast and simple FIA-chemiluminescence method for the evaluation of Roselle flowers as scavenger of the free radicals generated by UV irradiated antibiotics.

This work proposes a new method for the in vitro evaluation of the effect of UV irradiation on the production of free radicals and other reactive species during the photodecomposition of drugs. The method was based on the UV irradiation of antibiotics molecules to generate excited states that undergo to homolytic bond cleavages. These reactive species can be detected by their ability to oxidize the luminol, producing the electronically excited aminophtalate, which decays to the ground state releasing electromagnetic radiation in the visible zone of the spectrum. This method was applied to penicillin G, nafcillin, azlocillin and neomycin dissolved in water. It was found that the intensity of the luminol chemiluminescence emission (CL) was proportional to the concentration and dependent on the molecular structure of these drugs. Under the optimized conditions, it was found that penicillin and azlocillin were the most susceptible to photodegradation, while neomycin sulfate was the less affected by the UV light. It was observed that the addition to the antibiotics dissolutions of a hydro-alcoholic extract of petals of calyxes of Roselle reduced the CL intensity, indicating that the extract was able to scavenge the free radicals in the irradiated drugs. This result suggest that its addition to the antibiotics can help in the protection against the radicals formed during the exposition to solar light of patients treated with topic similar antibiotics.

Evaluation of neomycin analogues for HIV-1 RRE RNA recognition identifies enhanced activity simplified neamine analogues.

Synthetic neamine mimetics have been evaluated for binding to the HIV-1 Rev response element. Modified neamine derivatives, obtained from reductive amination of neamine, led to identification of new 6-amino modified neamine-type ligands with HIV-1 RRE binding affinity up to 20× that of neamine and up to 6× that of the more complex neomycin itself. This provides a noteworthy structure-activity increase and a useful lead to simplified, chemically accessible mimetics.

Building of neomycin-nucleobase-amino acid conjugates for the inhibition of oncogenic miRNAs biogenesis.

MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers, thus being oncogenic. The inhibition of oncogenic miRNAs (defined as the blocking of miRNAs' production or function) would find application in the therapy of different types of cancer in which these miRNAs are implicated. In this work, we describe the design and synthesis of new small-molecule RNA ligands with the aim of inhibiting Dicer-mediated processing of oncogenic miRNAs. One of the synthesized compound (4b) composed of the aminoglycoside neomycin conjugated to an artificial nucleobase and to amino acid histidine is able to selectively decrease miR-372 levels in gastric adenocarcinoma (AGS) cells and to restore the expression of the target LATS2 protein. This activity led to the inhibition of proliferation of these cells. The study of the interactions of 4b with pre-miR-372 allowed for the elucidation of the molecular mechanism of the conjugate, thus leading to new perspectives for the design of future inhibitors.

Efficient loading of ophthalmic drugs with poor loadability into contact lenses using functional comonomers.

Ophthalmic formulations have classically been administered to eyes through eye drops, which have poor delivery efficiency and require frequent instillation. As a means of overcoming these drawbacks, ocular-drug delivery via therapeutic contact lenses has caused great interest. A simple way to prepare therapeutic contact lenses is to immerse lenses in concentrated drug solution. However, not all ocular drugs can be efficiently loaded into contact lenses by a simple soaking. In particular, our previous study showed that two antibacterial ocular drugs, ofloxacin (OFX) and neomycin (NEO), were poorly loaded to poly(2-hydroxyethyl methacrylate) (pHEMA)-based contact lenses. Herein, we investigated whether alteration of lens composition using several negatively charged comonomers can enhance loading of ocular drugs with poor loadability (OFX and NEO). As comonomers, methacrylic acid (MAA), acrylic acid (AA), and 4-methyl-4-pentenoic acid (MPA) were used, generating p(HEMA-co-MAA), p(HEMA-co-AA), and p(HEMA-co-MPA) hydrogel-based contact lenses, respectively. Contact lenses containing comonomers exhibited an increase in loading of OFX and NEO. In particular, compared with pHEMA contact lenses, contact lenses containing 2.5 mol% AA exhibited enhanced loading of OFX and NEO, 18 and 53 times, respectively. Charge interactions between comonomers and the drug were considered primary factors in the substantial increase in drug loading.