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Mycobacterium tuberculosis ( Mtb ) contains potential G-quadruplex (PGQ) motifs in the genes espK and cyp51 , which are crucial for the bacteria's virulence within host cells. Aminoglycoside molecules are commonly used as antibiotics for ribosomal targets. This study provides insight into the interactions between these aminoglycosides and Mtb-PGQ sequences ( espK and cyp51 ), shedding light on the structural and thermodynamic dynamics of their binding. This study demonstrates the stability, affinity, and conformation of Mtb-PGQ in the presence of neomycin and streptomycin. Ultraviolet-visible spectroscopy (UV-vis), circular dichroism spectroscopy (CD), CD thermal melting, isothermal titration calorimetry (ITC), and fluorescence intercalator displacement (FID) assays were used to comprehensively examine these interactions. Our results reveal that neomycin with Mtb-PGQ exhibits hypochromism accompanied by a 4-5 nm red shift in the UV-visible absorption titration, whereas streptomycin exhibits a hypochromic shift without changes in the maximum wavelength. Notably, neomycin shows a nonlinear binding isotherm, suggesting the involvement of more than one binding process in the formation of neomycin. Mtb-PGQ complexes. Scatchard plot analysis indicates higher binding affinity values for neomycin compared with weaker affinity of streptomycin. CD studies reveal that neomycin decreases the ellipticity of Mtb-PGQ with a red shift while retaining the parallel topology, ultimately enhancing the thermal stability of both espK and cyp51 . In contrast, streptomycin destabilizes the cells. ITC analysis reveals that neomycin exhibits the strongest binding affinity for cyp51 , with the relative order being NEO- cyp51 > NEO- espk > STR- cyp51 > STR- espk. Moreover, thermodynamic analysis reveals that neomycin possesses a unique dual mode of binding through grooves as well as stacking. FID studies further confirm a lower DC 50 value for neomycin than for streptomycin, suggesting that neomycin is a strong displacer of thiazole orange. Thus, the results show that neomycin with amino groups selectively recognizes the grooves of cyp51 over espK .

The ongoing search for small molecule drugs that target ribonucleic acids (RNA) is complicated by a limited understanding of the principles that govern RNA-small molecule interactions. Here we have used stoichiometry-resolved native top-down mass spectrometry (MS) to study the binding of neomycin B to small model hairpin RNAs, an unstructured RNA, and a viral RNA construct. For 15-22 nt model RNAs with hairpin structure, we found that neomycin B binding to hairpin loops relies on interactions with both the nucleobases and the 2'-OH groups, and that a simple 5' or 3' overhang can introduce an additional binding motif. For a 47 nt RNA construct derived from stem IA of the human immunodeficiency virus 1 (HIV-1) rev response element (RRE) RNA, native top-down MS identified four different binding motifs, of which the purine-rich internal loop showed the highest affinity for neomycin B. Stoichiometry-resolved binding site mapping by native top-down MS allows for a new perspective on binding specificity, and has the potential to reveal unexpected principles of small molecule binding to RNA., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Staphylococcus aureus is a pathogen widely involved in wound infection due to its ability to release several virulence factors that impair the skin healing process, as well as its mechanism of drug resistance. Herein, sodium alginate and chitosan were combined to produce a hydrogel for topical delivery of neomycin to combat S. aureus associated with skin complications. The hydrogel was formulated by combining sodium alginate (50 mg/mL) and chitosan (50 mg/mL) solutions in a ratio of 9:1 (HBase). Neomycin was added to HBase to achieve a concentration of 0.4 mg/mL (HNeo). The incorporation of neomycin into the product was confirmed by scanning electron microscopy, FTIR and TGA analysis. The hydrogels produced are homogeneous, have a high swelling capacity, and show biocompatibility using erythrocytes and fibroblasts as models. The formulations showed physicochemical and pharmacological stability for 60 days at 4 ± 2 °C. HNeo totally inhibited the growth of S. aureus after 4 h. The antimicrobial effects were confirmed using ex vivo (porcine skin) and in vivo (murine) wound infection models. Furthermore, the HNeo-treated mice showed lower severity scores than those treated with HBase. Taken together, the obtained results present a new low-cost bioproduct with promising applications in treating infected wounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

3D multifunctional scaffold has been designed based on Cs/SA/NS/NPHA. Nanoparticles hydroxyapatite (NPHA) was prepared via precipitation method of sodium dihydrogen phosphate in presence calcium chloride. Different ratios of Chitosan (CS)/Sodium Alginate (SA) were used to prepare Cs/SA scaffolds in presence of CaCl 2 as a cross linker. NPHA was incorporated in CS/SA scaffold and neomycin sulfate (NS) was added as an antimicrobial agent. The structure and surface morphology of the scaffolds were investigated via infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM) and thermal gravimetric analysis (TGA) techniques. Additionally, Antimicrobial activity of the scaffold has evaluated against Gram- negative and Gram- positive bacteria. The result showed promising antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Furthermore, cytotoxicity against MG63 osteosarcoma cell and fibroblast normal cell line has investigated. The result showed anti-proliferative against MG63. DFT calculations and molecular docking were used to study the reactivity of the compounds. The results exhibited that Cs/SA/NS/NPHA is potent expected to be used in bone tissue regeneration., Competing Interests: Declaration of competing interest The author declares that there is no conflict to interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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, G 4 C 4 , in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of G 4 C 4 , with thermal enhancement decreasing with increasing pH and/or Na + concentration. Although G 4 C 4 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 G 4 C 4 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 G 4 C 4 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 NH 3 + groups participating in electrostatic interactions with G 4 C 4 . 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.

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., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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 IC 50 s., (© 2022 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

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., (© 2022 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

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.

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).

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 (P kasO * - neoN - metK -P kasO * - 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.

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-Cu 2 O, which could not only enhance the efficacy of aminoglycoside antibiotics, but also eliminate their environmental pollution by photocatalytic degradation. It was found that Cu 2 O 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 Cu 2 O 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 Cu 2 O facilitates the entry of antibiotics into cells is the reason for their synergy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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 tuberculosisH 37 RV (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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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)/H 2 O 2 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/H 2 O 2 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.

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., (Copyright © 2018. Published by Elsevier B.V.)

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., (Copyright © 2018. Published by Elsevier Ltd.)

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.

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.

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.

Aminoglycoside ototoxicity results in permanent loss of the sensory hair cells in the mammalian cochlea. It usually begins at the basal turn causing high-frequency hearing loss. Here we describe previously unreported resistance of hair cells to neomycin ototoxicity in the extreme basal (hook) region of the developing cochlea of the C57BL/6 mouse. Organ of Corti explants from mice at postnatal day 3 were incubated (37 °C, 5% CO 2 ) in normal culture medium for 19.5 h prior to and after exposure to neomycin (1 mM, 3 h). To study neomycin uptake in the hair cells, cochlear explants were incubated with Neomycin Texas-red (NTR) conjugate. As expected, exposure to neomycin significantly reduced the survival of inner (IHC) and outer hair cells (OHC). IHC survival rate was high in the apical segment and low in the basal segment. OHC were well preserved in the apical and hook regions, with substantial OHC loss in the basal segment. The NTR uptake study demonstrated that the high survival rate in the extreme basal turn OHC was associated with low NTR uptake. Treatment with a calcium chelator (BAPTA), which disrupts the opening of mechanoelectrical (MET) transduction channels, abolished or reduced NTR uptake in the hair cells throughout the cochlea. This confirmed the essential role of MET channels in neomycin uptake and implied that the transduction channels could be impaired in the hook region of the developing mouse cochlea, possibly as a result of the cadherin 23 mutation responsible for the progressive deafness in C57BL/6 mice.

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.

A new analytical method was developed to detect neomycin in complex biological samples using molecularly imprinted polymer to construct an optical sensor. Fluorescent neomycin-imprinted polymers (fMIPs) containing both imprinted cavity and boronate affinity site were synthesized on the surface of silica-modified quantum dots. The fMIPs exhibited high selectivity to neomycin by having two binding sites for the target analyte. Neomycin analogues (competing for imprinted cavity) and D-glucose (competing for the boronate affinity site) did not affect the selectivity of the fMIPs. When combined with a fluorescent microplate reader, the obtained fMIP sensor displayed a linear concentration-dependent fluorescence quenching in response to neomycin in the range of 2-1000 μg/L, with a limit of detection as 0.16 μg/L. The fMIP sensor was able to detect trace neomycin in biological samples accurately after simple sample pre-treatment. The sensitivity of the fMIP sensor was higher than HPLC equipped with a fluorescence detector. The fMIP sensor containing the doubly selective binding sites provides a selective, sensitive, accurate, and high through-put approach for neomycin monitoring., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aminoglycoside antibiotics, discovered as natural products in the 1940s, demonstrate a broad antimicrobial spectrum. Due to their nephrotoxic and ototoxic side effects, however, their widespread clinical usage has typically been limited to the treatment of serious infections. Neomycin B, first isolated from strains of Streptomyces in 1948, is one such drug that was approved for human use by the U.S. Food and Drug Administration in 1964. Only within the last 11 years has the biochemical pathway for its production been elaborated, however. Here we present the three-dimensional architecture of NeoB from Streptomyces fradiae, which is a pyridoxal 5'-phosphate or PLP-dependent aminotransferase that functions on two different substrates in neomycin B biosynthesis. For this investigation, four high resolution X-ray structures of NeoB were determined in various complexed states. The overall fold of NeoB is that typically observed for members of the "aspartate aminotransferase" family with the exception of an additional three-stranded antiparallel β-sheet that forms part of the subunit-subunit interface of the dimer. The manner in which the active site of NeoB accommodates quite different substrates has been defined by this investigation. In addition, during the course of this study, we also determined the structure of the aminotransferase GenB1 to high resolution. GenB1 functions as an aminotransferase in gentamicin biosynthesis. Taken together, the structures of NeoB and GenB1, presented here, provide the first detailed descriptions of aminotransferases that specifically function on aldehyde moieties in aminoglycoside biosynthesis., (© 2018 The Protein Society.)

Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre-loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/μL when compared to 2 and 100 ng/μL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/μL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.

We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses. Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS), virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced agglutination of viral particles, the high capture efficiency and affinity constant indicate that this neoprotein is comparable to natural glycosylated mucin, suggesting that this material could potentially be used as anti-infective barriers to prevent virus from invading host cells. The study also rationalizes the feasibility of antiviral drug development based on OA or other antiviral small molecules conjugated protein strategies., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein-phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P 2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH-PI(4,5)P 2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH-PI(4,5)P 2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P 2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein-phosphoinositide interactions.

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 10 3 /1.44 × 10 3  L mol -1 at 291 K. The values of ΔH θ , ΔS θ , and ΔG θ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern-Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA-AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS-DNA-AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A-T rich region of DNA.

Silane crosslinked biopolymer based novel pH-responsive hydrogels were fabricated by blending the cationic (chitosan) and anionic (alginate) polymers with poly(vinyl alcohol). Tetraethoxysilane (TEOS) was used, as a crosslinker in different amounts due to its nonhazardous nature, to study its impact on physical and chemical properties of the prepared injectable hydrogels along with the controlled release of drug. The swelling response of the prepared hydrogels was examined in different solvent media which exhibited decreased swelling ratio with increase in the amount of TEOS. All the fabricated hydrogels represented highest swelling at acidic pH while low swelling at basic and neutral pH. This specific pH sensitive behavior at pH 7 made them an appropriate candidate for the injectable controlled drug delivery in which Neomycin Sulfate (NMS) was successfully loaded on suitable hydrogel (comprising 50μL TEOS) to study its release mechanism. The results revealed that in simulated gastric fluid (SGF), hydrogel released the entire drug (NMS) in initial 30min while in simulated intestinal fluid (SIF), NMS was released in a controlled way up to 83% in 80min. These results endorsed that the hydrogels could be practiced as a smart intelligent material for injectable controlled drug delivery as well as for other biomedical applications at physiological pH., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Specific detection of pathogens has long been recognized as a vital strategy in the control of infectious diseases. Two novel theranostic neomycin analogs exhibit efficient targeting, labelling and killing of broad spectrum bacteria while not damaging macrophage-like cells. Furthermore, lipidated probe 2 clearly showed antibacterial activity against methicillin-resistant S. aureus.

We present a colorimetric method for the detection of aminoglycoside antibiotics such as neomycin (NEO) using a reddish anionic dye, amaranth (AR 3- ). Under acidic conditions, at which NEO exists in fully protonated form (NEOH 6+ ), the AR 3- anion associates with the NEOH 6+ cation to form a precipitate, NEOH(AR) 2 . The precipitate was soluble in a buffer solution of pH 8.5, yielding a reddish solution with an absorption maximum at around 520 nm. Tobramycin and gentamycin, which exist as pentavalent cations under acidic conditions, gave almost the same results. On the other hand, kanamycin, amikacin and streptomycin, which would exist as tri- and tetravalent cations, were not precipitated. Thus, the AR 3- anion could be considered to be an analytical reagent for specific aminoglycosides with polycationic functionality. However, since the precipitation reaction was considerably affected by other anions, a separation method using the tetraphenylborate anion was employed as a pretreatment. The separation method involves precipitating the polycationic aminoglycosides with the tetraphenylborate anion, washing the precipitate with acetonitrile, and re-precipitating the aminoglycosides as hydrochloride salts. Thus, the present method was applied to a microtiter plate assay of the products in an NEO-producing culture broth.

The present study demonstrates the use of self-assembled nanostructures of cationic amphiphilic azobenzene-neomycin (a small molecule) conjugate, Azo-Neo, as delivery vector for plasmid DNA. These nanostructures efficiently condensed nucleic acid and formed more compact nanoassemblies. DLS analysis showed size and zeta potential of the resulting Azo-Neo/pDNA nanoassemblies ∼153.7nm and +7.26mV, respectively. The nanoassemblies were characterized by physicochemical techniques and evaluated for its toxicity and ability to deliver nucleic acid therapeutics. The flow cytometry results on MCF-7 and HEK293T cells revealed that Azo-Neo/pDNA nanoassemblies transfected ∼31% and 23% cells, respectively, at a w/w ratio of 250, while the standard transfection reagent, bPEI/pDNA complex, could transfect only ∼21% and 29% cells, respectively, at its best w:w ratio of 2.3. MTT and hemolysis assays showed the non-toxic nature of the projected nanoassemblies and nanostructures, respectively, at various concentrations. Further, Azo-Neo nanostructures showed efficient antibacterial activity against different strains, laboratory strain of Staphylococcus aureus (MTCC 740) as well as MRSA strains (Staphylococcus aureus ATCC 33591, ATCC 43300 and ATCC 700699). These results ensure the great potential of these nanostructures in gene delivery and antimicrobial applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To develop a novel neomycin sulfate-loaded hydrogel dressing (HD), numerous neomycin sulfate-loaded HDs were prepared with various amounts of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and sodium alginate (SA) using freeze-thawing technique, and their physical dressing properties, drug release, in vivo wound curing and histopathology in diabetic-induced rats were assessed. SA had a positive effect on a swelling capacity, but a negative effect on the physical dressing properties and drug release of HD. However, PVP did the opposite. In particular, the neomycin sulfate-loaded HD composed of drug, PVA, PVP and SA at the weight ratio of 1/10/0.8/0.8 had excellent swelling and bioadhesive capacity, good elasticity and fast drug release. Moreover, this HD gave more improved wound curing effect compared to the commercial product, ensured the disappearance of granulation tissue and recovered the wound tissue to normal. Therefore, this novel neomycin sulfate-loaded HD could be an effective pharmaceutical product for the treatment of wounds.

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 and that the inhibition of these oncogenic miRNAs could find application in the therapy of different types of cancer. Herein, we describe the synthesis and biological evaluation of new small-molecule drugs that target oncogenic miRNAs production. In particular, we chose to target two miRNAs (i.e., miRNA-372 and -373) implicated in various types of cancer, such as gastric cancer. Their precursors (pre-miRNAs) are overexpressed in cancer cells and lead to mature miRNAs after cleavage of their stem-loop structure by the enzyme Dicer in the cytoplasm. Some of the newly synthesized conjugates can inhibit Dicer processing of the targeted pre-miRNAs in vitro with increased efficacy relative to our previous results (D.D. Vo et al., ACS Chem. Biol. 2014, 9, 711-721) and, more importantly, to inhibit proliferations of adenocarcinoma gastric cancer (AGS) cells overexpressing these miRNAs, thus representing promising leads for future drug development., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Acanthamoeba keratitis is a serious infection with blinding consequences and often associated with contact lens wear. Early diagnosis, followed by aggressive topical application of drugs, is a prerequisite in successful treatment, but even then prognosis remains poor. Several drugs have shown promise, including chlorhexidine gluconate; however, host cell toxicity at physiologically relevant concentrations remains a challenge. Nanoparticles, subcolloidal structures ranging in size from 10 to 100 nm, are effective drug carriers for enhancing drug potency. The overall aim of the present study was to determine whether conjugation with gold nanoparticles enhances the antiacanthamoebic potential of chlorhexidine. Gold-conjugated chlorhexidine nanoparticles were synthesized. Briefly, gold solution was mixed with chlorhexidine and reduced by adding sodium borohydride, resulting in an intense deep red color, indicative of colloidal gold-conjugated chlorhexidine nanoparticles. The synthesis was confirmed using UV-visible spectrophotometry that shows a plasmon resonance peak of 500 to 550 nm, indicative of gold nanoparticles. Further characterization using matrix-assisted laser desorption ionization-mass spectrometry showed a gold-conjugated chlorhexidine complex at m/z 699 ranging in size from 20 to 100 nm, as determined using atomic force microscopy. To determine the amoebicidal and amoebistatic effects, amoebae were incubated with gold-conjugated chlorhexidine nanoparticles. For controls, amoebae also were incubated with gold and silver nanoparticles alone, chlorhexidine alone, neomycin-conjugated nanoparticles, and neomycin alone. The findings showed that gold-conjugated chlorhexidine nanoparticles exhibited significant amoebicidal and amoebistatic effects at 5 μM. Amoebicidal effects were observed by parasite viability testing using a Trypan blue exclusion assay and flow-cytometric analysis using propidium iodide, while amoebistatic effects were observed using growth assays. In contrast, chlorhexidine alone, at a similar concentration, showed limited effects. Notably, neomycin alone or conjugated with nanoparticles did not show amoebicidal or amoebistatic effects. Pretreatment of A. castellanii with gold-conjugated chlorhexidine nanoparticles reduced amoeba-mediated host cell cytotoxicity from 90% to 40% at 5 μM. In contrast, chlorhexidine alone, at similar concentrations, had no protective effects for the host cells. Similarly, amoebae treated with neomycin alone or neomycin-conjugated nanoparticles showed no protective effects. Overall, these findings suggest that gold-conjugated chlorhexidine nanoparticles hold promise in the improved treatment of A. castellanii keratitis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neomycin-conjugated homopyrimidine oligo 2'-deoxyribonucleotides have been synthesized on a solid phase and their potential as triplex forming oligonucleotides (TFOs) with DNA-duplexes has been studied. For the synthesis of the conjugates, C-5, C-2' and C-4'-tethered alkyne-modified nucleoside derivatives were used as an integral part of the standard automated oligonucleotide chain elongation. An azide-derived neomycin was then conjugated to the incorporated terminal alkynes by Cu(I)-catalyzed 1,3-dipolar cycloaddition (the click chemistry). Concentrated ammonia released the desired conjugates in acceptable purity and yields. The site of conjugation was expectedly important for the Hoogsteen-face recognition: C-5-conjugation showed a notable positive effect, whereas the influence of the C-2' and C-4'-modification remained marginal. In addition to conventional characterization methods (UV- and CD-spectroscopy), (19)F NMR spectroscopy was applied for the monitoring of triplex/duplex/single strand-conversions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The success of drug delivery depends on the efficiency of the route of administration, which in turn relies on properties of the drug and its transport vehicle. A quantitative knowledge of association of drugs with transport vehicles is lacking when the latter are in the category of self assembled structures. The work reported in this manuscript addresses the mechanism of partitioning of naproxen, diclofenac sodium, neomycin and lincomycin in the micelles of hexadecytrimethylammonium bromide and that is quantitatively based on the measurement of thermodynamic parameters of interactions by using isothermal titration calorimetry. The addressed mechanism of partitioning is based on the identification of the type of interactions of these drugs with the surfactant micelles and monomers, along with the effect of the former on the micellization properties of the surfactant. The conclusions are based on the interpretation of the values of partitioning constant, standard molar enthalpy change, standard molar entropy change and the stoichiometry of the interaction. The results of this study have implications for deriving guidelines for the target oriented synthesis of new drugs that are to be used for effective delivery via micellar media., (Copyright © 2015 Elsevier B.V. All rights reserved.)

DNA-protein recognition has shown us the importance of DNA shapes in the recognition process. Specific high-affinity targeting of DNA shapes by small molecules is desirable for many biological applications that involve regulation of DNA based processes. Here, the effect of linker length and rigidity on the affinity of a conjugated neomycin dimer for a specific DNA shape (B* form) AT-rich DNA was explored. Binding constants approximating 10(8)M(-1) for optimal linker lengths of 18-19 atoms are reported herein., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

By the forced unfolding of polyglutamine and polyalanine homopeptides in competing α-helix and β-hairpin secondary structures, we disentangle equilibrium free energetics from nonequilibrium dissipative effects. We find that α-helices are characterized by larger friction or dissipation upon unfolding, regardless of whether they are free energetically preferred over β-hairpins or not. Our analysis, based on MD simulations for atomistic peptide models with explicit water, suggests that this difference is related to the internal friction and mostly caused by the different number of intrapeptide hydrogen bonds in the α-helix and β-hairpin states.

The carboxylic groups of glutamic acid and aspartic acid residues of catalase (CAT) were chemically modified using the treatment of the enzyme with 1-ethyl-3-(3'-dimethylamino) carbodiimide hydrochloride (EDC) and neomycin. The effect of covalent attachment of neomycin on the enzymatic activity, conformational and aggregation properties of CAT was investigated. The modification of CAT with different concentrations of neomycin showed two different types of behavior, depending up on the concentration range of neomycin. In the concentration range from 0.0 to 5.2 mM, neomycin-modified CAT, compared to the native enzyme exhibited higher a-helix content, reduced surface hydrophobicity, little enhancement in CAT activity and a better protection against thermal aggregation, whereas at concentrations greater than 5.2 mM, the modified enzyme exhibited a significant decrease in CAT activity and an increase in random coil content which may result in disorder in the protein structure and increase in thermal aggregation. This modification is a rapid and simple approach to investigate the role of aspartate and glutamate residues in the structure, function and folding of CAT.

RNA interference (RNAi) is a promising tool to regulate gene expression by external double stranded RNAs (dsRNAs) such as siRNAs. As an efficient method to deliver siRNAs to liver cells, we propose a novel strategy using vitamin E (VE)-conjugated neomycin derivatives. With the aim of delivering RNAi-based drugs to liver cells, several tripod-type and prodrug-type neomycin derivatives were synthesized, all of which were thermodynamically stabilized RNA duplexes. The prodrug-type derivative 7 and the tripod-type derivative 10 were delivered to liver cancer cells and successfully induced RNAi activity. These results indicated the potential use of natural aminoglycosides as carriers of RNAi drugs., (Copyright © 2015. Published by Elsevier Ltd.)