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.     

Synthesis and evaluation of novel neamine derivatives effectively targeting to RNA

Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of these derivatives to A site of 16S RNA and TAR RNA indicate that the modification on 5-hydroxyl of neamine by amino acid can enhance the binding affinity of neamine. Compound 9 (NEL) shows some antibacterial activities. These results demonstrate that modification on 5-hydroxyl group of neamine may provide a promising way for the development of potential candidates effectively targeting to RNAs.     

Antimicrobial activity of amphiphilic neamine derivatives: Understanding the mechanism of action on Gram-positive bacteria

Amphiphilic aminoglycoside derivatives are potential new antimicrobial agents mostly developed to fight resistant bacteria. The mechanism of action of the 3′,6-dinonyl neamine, one of the most promising derivative, has been investigated on Gram-negative bacteria, including P. aeruginosa. In this study, we have assessed its mechanism of action against Gram-positive bacteria, S. aureus and B. subtilis. By conducting time killing experiments, we assessed the bactericidal effect induced by 3′,6-dinonyl neamine on S. aureus MSSA and MRSA. By measuring the displacement of BODIPY?-TR cadaverine bound to lipoteichoic acids (LTA), we showed that 3′,6-dinonyl neamine interacts with these bacterial surface components. We also highlighted the ability of 3′,6-dinonyl neamine to enhance membrane depolarization and induce membrane permeability, by using fluorescent probes, DiSC₃C(5) and propidium iodide, respectively. These effects are observed for both MSSA and MRSA S. aureus as well as for B. subtilis. By electronic microscopy, we imaged the disruption of membrane integrity of the bacterial cell wall and by fluorescence microscopy, we demonstrated changes in the localization of lipids from the enriched-septum region and the impairment of the formation of septum. At a glance, we demonstrated that 3′,6-dinonyl neamine interferes with multiple targets suggesting a low ability of bacteria to acquire resistance to this agent. In turn, the amphiphilic neamine derivatives are promising candidates for development as novel multitarget therapeutic antibiotics.     

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.     

Rational design and synthesis of potent aminoglycoside antibiotics against resistant bacterial strains

Based on the structural information of biomacromolecule-aminoglycoside complexes, a series of kanamycin B analogues were rationally designed and synthesized. A convenient approach to the construction of kanamycin derivatives, in which the C4′-position on ring I of neamine moiety was modified, was developed. Most synthetic analogues exhibited good to excellent antibiotic activity against some typical drug-resistant bacteria. The disclosed results suggested that the C4′-position of aminoglycosides such as kanamycin may be an ideal site for modification to gain new modifying enzyme-resistant aminoglycoside antibiotics.     

Studies on the synthesis of neamine-dinucleosides and neamine-PNA conjugates and their interaction with RNA

Two types of neamine derivatives, neamine-dinucleotide conjugates 8a-g and neamine-PNA conjugates 12a-c and 14a-d, were synthesized. Compound 8a-g were synthesized by the condensation of azido-neamine with dinucleotide-5'-carboxylic acids, followed by reduction and deprotection. Compound 12a-c and 14a-d were synthesized by the similar strategy. The binding affinities of conjugates 8a-g, 12a-c, and 14a-d towards 16S RNA, 18S RNA, and TAR RNA were evaluated by SPR. It indicates that conjugates 12a-c and 14a-d interact with 16S, 18S RNA at the same level as that of neamine, 14a and 14d show about twofold binding affinities to TAR RNA compared to that of neamine. However, the neamine-dinucleotide conjugates 8a-g exhibit very weak binding affinities to 16S, 18S, and TAR RNA, computer modelling results that negative-negative electrostatic repulsion of phosphate group in compound 8a-g and RNA leads to a sharp decrease of the binding affinities compared with that of neamine, neamine-nucleoside and neamine-PNA conjugates.     

Synthesis and antibacterial activity of novel neamine derivatives

Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-3′,4′-dideoxyneamine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative showed effective activity against Staphylococcus aureus expressing a bifunctional aminoglycoside-modifying enzyme AAC(6′)-APH(2″).     

Selectively guanidinylated derivatives of neamine. Syntheses and inhibition of anthrax lethal factor protease

A series of mono-, di-, and tri-guanidinylated derivatives of neamine were prepared via selective guanidinylation of neamine. These molecules represent a novel scaffold as inhibitors of anthrax lethal factor zinc metalloprotease. Methods for the synthesis of these compounds are described, and structure-activity relationships among the series are analyzed. In addition, initial findings regarding the mechanism of LF inhibition for these molecules are presented.     

Study of aminoglycoside-nucleic acid interactions by an HPLC method

The interactions of a number of aminoglycoside antibiotics with tRNA and DNA were studied by an HPLC method. based on tRNA and DNA peak size exclusion. Among the compounds studied (deoxystreptamine, neamine, neomycin B, kanamycin A, gentamicin A, netilmicin, streptomycin, and the synthetic neamine analogue BKN3), neomycin B and the synthetic analogue of neamine were proved to be the most potent binders.     

A new method for selective N-acylation of aminoglycoside antibiotics

Per-N-formylation of aminoglycoside (aminocyclitol) antibiotics followed by mild hydrolysis with aqueous ammonia gave mono-N-deformylated derivatives. Each positional isomer of the mono-N-deformylated derivatives thus obtained was separated by column chromatography on Amberlite CG-50 (NH₄⁺ ). Acylation of mono-N-deformylated derivatives gave the corresponding mono-N-acylated derivatives. The N-formyl groups of the mono-N-acylates were removed by the treatment with dilute aqueous hydrazine acetate, whereas the newly introduced N-acyl group was stable under these conditions. The 1-N-formyl group of the deoxystreptamine moiety of per-N-formylated aminoglycoside antibiotics containing neamine (or 3′-deoxyneamine) is more readily deformylated than the 3-N-formyl group. In this report, isolation and structural-elucidation studies, including ¹³C-n.m.r. spectral assignments, of positional isomers of tri-N-formyl derivatives of xylostasin (1), 3′-deoxyxylostasin (2), kanamycin A (3), and neamine (4) are described. This selective N-acylation provides a useful method for the preparation of 1-N-modified derivatives, and the synthesis of 3′-deoxybutirosin A (2f) from 2 is described in detail as an example.     

Chemical modification of aminocyclitol antibiotics

The aminocyclitol antibiotic neamine has been chemically modified at the hydroxyl group on C-6 of the 2-deoxystreptamine moiety. The partially acetylated neamine derivatives, 6,3′,4′-tri-O-acetyl- (3) and 5,3′,4′-tri-O-acetyl-1,3,2′,6′-tetra-N-(ethoxycarbonyl)neamine (4), were prepared by random hydrolysis of the 5,6-O-ethoxyethylidene derivative (2), followed by chromatographic purification. Condensation of 4 and 2,3,5-tri-O-benzoyl-d-ribofuranosyl chloride led to 6-O-(β-d-ribofuranosyl)neamine (7). Analogous condensation of 4 with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide afforded the corresponding 6-O-(d-hexopyranosyl)neamines.     

Separation and Quantitation of Neomycins as Free Base by Paper Chromatography

Neomycin B and C and neamine were separated as free bases in the solvent system methyl ethyl ketone-tert-butyl alcohol-methanol-6.5 N NH₄OH (16:3:1:6 v/v) and quantitatively estimated.     

Mechanism of RNA cleavage catalyzed by sequence specific polyamide nucleic acid-neamine conjugate

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.     

新霉胺通过抑制血管生成素活性抑制人黑色素瘤细胞增殖、迁移和侵润

新霉胺（neamine）是一种无毒的新霉素（neomycin）降解产物;已有研究证明,其可抑制血管生成素（angiogenin,ANG）诱导的内皮细胞血管生成作用,阻滞异种移植的人结肠腺癌在裸鼠的生长.本研究证明,新霉胺对人黑色瘤细胞株A375的细胞增殖、迁移和侵润作用.MTT法及软琼脂培养显示,新霉胺可明显抑制A375细胞的增殖和集落形成能力. Transwell试验证明,新霉胺可阻滞A375细胞,乃至血管生成素诱导的A375细胞的迁移和侵润能力.此外,免疫荧光揭示新霉胺可阻断血管生成素的核转位,从而抑制血管生成素诱导的A375细胞增殖.上述结果提示,新霉胺可通过抑制血管生成素的核转位,从而抑制肿瘤细胞增殖、迁移和侵润.鉴于与新霉素比较,新霉胺毒性小,因此新霉胺可望作为黑色素瘤治疗的先导药物,颇具开发前景和潜力.     

Synthesis of fluorescent and photoaffinity-labeled derivatives of bisphenol A and their inhibitory activity toward hypoxic expression of erythropoietin

Bisphenol A derivatives, possessing a fluorescent dye and a photo-reactive group, were synthesized from bisphenol A, and the inhibitory activity of the derivatives was evaluated against hypoxic response. The synthesized derivatives were found to inhibit the hypoxic expression of erythropoietin in Hep3B cells as well as bisphenol A.     

Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5′UNNNC3′ loops for the kanamycin A derivative (where N is any nucleotide); 5′UNNC3′ loops for the tobramycin derivative; 5′UNC3′ loops for the neamine derivative; and 5′UNNG3′ loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have K_d values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.     

Neamine derivatives having a nucleobase with a lysine or an arginine as a linker, their synthesis and evaluation as potential inhibitors for HIV TAR-Tat

Neamine derivatives bearing a nucleobase, adenine, cytosine, guanine or thymine with a lysine or an arginine as a linker have been synthesized and its potential as the inhibitor for HIV TAR-Tat interaction examined. Among them, modified neamine having an arginine-nucleobase showed a higher inhibition than that of the one having a lysine-nucleobase. The difference of the nucleobase anchor did not characterize inhibition specificity. Also, stereochemistry of the amino acid in the compounds causes no difference in the inhibition for TAR-Tat.     

NMR and amber analysis of the neamine pharmacophore for the design of novel aminoglycoside antibiotics

The dependence of the solution structure of neamine on pH was determined by NMR and AMBER molecular dynamics methods at pD 3.3, pD 6.5, and pD 7.4 in D₂O at 25 °C. Unlike neamine structures at pD 3.3 and 6.5, which essentially showed only one conformer, slowly exchanging primary, P-state, and secondary, S-state, neamine conformers populated on the NMR time scale at ∼80% and ∼20%, respectively, were detected at pD 7.4 with kinetic constants k_on(P→S) = 1.9771 s⁻¹ and k_off(S→P) = 1.1319 s⁻¹. A tertiary, T-state, neamine species populated at ∼3% was also detected by NMR at pD 7.4. The pKa values determined by NMR titration experiments are pKa1 6.44 ± 0.13 for N3 of ring-II, pKa2 7.23 ± 0.09 for N2′ of ring-I, pKa3 7.77 ± 0.19 for N1 of ring-II, and pKa4 8.08 ± 0.15 for N6′ of ring-I. Ring-I and ring-II of the P-state neamine and ring-I of the S and T-states of neamine possess the ⁴C₁ chair conformation between pD 3.3 and pD = 7.4. In contrast, ring-II of the S and T-states of neamine most likely adopt the ⁶rH₁ half-chair conformation. The P and S-states of neamine exhibit a negative syn-ψ glycosidic geometry. The exocyclic aminomethyl group of ring-I adopts the gt exocyclic rotamer conformation around physiological pHs while the gg exocyclic rotamer conformation predominates in acidic solutions near and below pH 4.5. Neamine exists in the P-state as a mixture of tetra-/tri-/di-protonated species between pD 4.5 and pD 7.4, while the S-state neamine exist only in a di-protonated species around physiological pDs. The existence of the S-state neamine may facilitate binding of neamine-like aminoglycosides by favorable entropy of binding to the A-site of 16S ribosomal RNA, suggesting that novel aminoglycoside compounds carrying a S-state neamine pharmacophore can be developed.     

Isolation and characteristics of the biological and genetic properties of neamin-resistant mutants of Salmonella typhimurium and Salmonella dublin candidates for vaccine creation

Mutants, resistant to neamine and spectinomycin, have been isolated from S. typhimurium and S. dublin highly virulent strains. The neamine-resistant mutants can be divided into 3 classes in accordance with their sensitivity to streptomycin: sensitive, resistant to low and high concentrations of this antibiotic. The transduction analysis with the use of bacteriophage P 22 has revealed that the spectinomycin-resistant mutations under study are spc A mutations, while the mutations leading to resistance to neamine in class Near Strr 500 are nea B mutations. The mutation leading to resistance to spectinomycin (spc A) has been found to produce no changes in the virulence of salmonellae in the intraperitoneal infection of mice. The mutations leading to resistance to neamine and streptomycin (nea B and str A) have been found to decrease virulence.