A novel 12-membered ring non-antibiotic macrolide EM982 attenuates cytokine production by inhibiting IKKß and IκBα phosphorylation.

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.

Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics.

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

Wound therapy via a photo-responsively antibacterial nano-graphene quantum dots conjugate.

Common bacterial pathogens have become resistant to traditional antibiotics, representing an indispensable public health crisis. Photodynamic therapy (PDT), especially when common visible light sources are used as photodynamic power, is a promising bactericidal method. Based on the special photodynamic properties triggered by commonly available light emitting diode (LED) lamps, a kind of graphene quantum dots (GQDs) based composite system (termed GQDs@hMSN(EM)) was prepared through loading both GQDs and erythromycin (EM) into the hollow mesoporous silica nanoparticle (hMSN), aiming to achieve joint antimicrobial effect. Bacterial density experiments confirmed that GQDs@hMSN(EM) had combined antimicrobial effects from photodynamic effect and drug release on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In animal models, the healing degree of wounds infected by bacteria also confirmed that GQDs@hMSN(EM) group had the best therapeutic effect, with the significantly reduced inflammatory factors in blood. Different from traditional GQDs synthesized by solvothermal method, the as-prepared GQDs@hMSN can produce singlet oxygen (1O2) under light exposure to destroy the structure of bacteria, thus achieving highly efficient antimicrobial effect. The GQDs@hMSN(EM) in this work possesses good antimicrobial activity, sufficient drug loading, and controllable drug release ability, which provides a new opportunity for GQDs-based nanoplatform to enhance antimicrobial effect and reduce their drug resistance.

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Characterization of four unknown impurities in azithromycin and erythromycin imino ether using two-dimensional liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance.

RATIONALE: A simple and sensitive method was developed for the separation and characterization of four unknown impurities in azithromycin and erythromycin imino ether using two-dimensional liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (2D LC/QTOFMS) with positive and negative electrospray ionization. METHODS: The chromatographic separation in the first dimension was performed with a Waters Xbridge RP18 column in gradient mode using binary mobile phase: (A) phosphate buffer (pH 8.2)-acetonitrile (47:53, v/v) and (B) water-acetonitrile (90:10, v/v). In the second dimension, the chromatographic separation was performed using a Shimadzu Shim-pack GISS C18 column with volatile mobile phases: (A) ammonium formate solution (10 mM) and (B) methanol. RESULTS: The molecular formulae and structures of the four impurities were deduced based on the LC/MS/MS data, and further confirmed using 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC and HMBC NMR spectra after semi-preparative isolation of impurities. In addition, the mechanism for the formation of the impurities was also proposed. CONCLUSIONS: The contradiction between the non-volatile salt mobile phase and mass spectrometry was solved by means of a multiple heart-cutting 2D LC approach and on-line desalination technology. Four impurities were separated and characterized. These results could further improve the method of official monographs in pharmacopoeias and guides to improve the process of reducing impurity content.

Comparing the release of erythromycin and vancomycin from calcium polyphosphate hydrogel using different drug loading methods.

Calcium polyphosphate (CPP) hydrogel is used to load erythromycin (EM) and vancomycin (VCM) by means of two loading methods: they are either added directly to the formed CPP hydrogel (Gel Mixture method) or mixed with CPP powders, followed by the formation of CPP-antibiotic hydrogel (Powder Mixture method). The release of loaded antibiotics from CPP hydrogel is measured up to 48 hr. Compared to Powder Mixture method, Gel Mixture method significantly reduced the burst release of embedded antibiotics. A significant change in CPP hydrogel Raman characteristic peaks is observed only in Gel Mixture method, indicating a close interaction between embedded antibiotics with CPP hydrogel matrix. In contrast, a similarity between characteristic peaks of CPP hydrogel and Powder Mixture method shows that antibiotic incorporation does not interfere with CPP gel formation, resulting in no ionic interaction between antibiotic and polyphosphate chains. Rheometer analysis further confirms that the hydrophobic nature of EM impacts the viscoelastic properties of CPP hydrogel, whereas the hydrophilic VCM exhibits a higher loading efficiency. The potential application of CPP hydrogel as a ceramic matrix for sustained drug release warrants further investigation.

High-resolution crystal structures of ribosome-bound chloramphenicol and erythromycin provide the ultimate basis for their competition.

The 70S ribosome is a major target for antibacterial drugs. Two of the classical antibiotics, chloramphenicol (CHL) and erythromycin (ERY), competitively bind to adjacent but separate sites on the bacterial ribosome: the catalytic peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), respectively. The previously reported competitive binding of CHL and ERY might be due either to a direct collision of the two drugs on the ribosome or due to a drug-induced allosteric effect. Because of the resolution limitations, the available structures of these antibiotics in complex with bacterial ribosomes do not allow us to discriminate between these two possible mechanisms. In this work, we have obtained two crystal structures of CHL and ERY in complex with the Thermus thermophilus 70S ribosome at a higher resolution (2.65 and 2.89 Å, respectively) allowing unambiguous placement of the drugs in the electron density maps. Our structures provide evidence of the direct collision of CHL and ERY on the ribosome, which rationalizes the observed competition between the two drugs.

Blue-light photoelectrochemical sensor based on nickel tetra-amined phthalocyanine-graphene oxide covalent compound for ultrasensitive detection of erythromycin.

In this study, we developed a novel photoelectrochemical (PEC) sensor for the highly sensitive detection of erythromycin by functionalising graphene oxide (GO) with nickel tetra-amined phthalocyanine (NiTAPc) through covalent bonding, which resulted in the formation of NiTAPc-Gr. The fabricated sensor showed a higher PEC efficiency under blue light, exhibiting a peak wavelength of 456 nm, as compared to that of the monomer. Further, the NiTAPc-Gr/indium tin oxide (ITO) sensor exhibited a photocurrent that was 50-fold higher than that for a GO/ITO sensor under the same conditions. Under optimal conditions, the NiTAPc-Gr PEC sensor showed a linear response for erythromycin concentrations ranging from 0.40 to 120.00 µmol L-1, with the minimum limit for detection being 0.08 µmol L-1. Thus, the NiTAPc-Gr sensor exhibited superior performance and excellent PEC characteristics, high stability, and good reproducibility with respect to the sensing of erythromycin. Moreover, it is convenient to use, fast, small, and cheap to produce. Hence, it should find wide use in the analysis of erythromycin in real-world applications.

Real-time homonuclear broadband decoupled pure shift COSY.

The present manuscript reports development and applications of real-time homonuclear broadband decoupled pure shift version of in-phase zero-quantum filtered COSY (PS-IPZF-COSY) and clean in-phase COSY (PS-CLIP-COSY) pulse schemes. In contrast to the conventional COSY schemes, these pure shift versions provide enhanced spectral resolution and simplify the chemical shift correlation analysis of scalar coupled spins in complex organic molecules, which are exemplified for erythromycin A, estradiol, and a mixture of estradiol and testosterone.

From Cell to Beak: In-Vitro and In-Vivo Characterization of Chicken Bitter Taste Thresholds.

Bitter taste elicits an aversive reaction, and is believed to protect against consuming poisons. Bitter molecules are detected by the Tas2r family of G-protein-coupled receptors, with a species-dependent number of subtypes. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors-ggTas2r1, ggTas2r2 and ggTas2r7. This minimalistic bitter taste system in chickens was used to determine relationships between in-vitro (measured in heterologous systems) and in-vivo (behavioral) detection thresholds. ggTas2r-selective ligands, nicotine (ggTas2r1), caffeine (ggTas2r2), erythromycin and (+)-catechin (ggTas2r7), and the Tas2r-promiscuous ligand quinine (all three ggTas2rs) were studied. Ligands of the same receptor had different in-vivo:in-vitro ratios, and the ggTas2r-promiscuous ligand did not exhibit lower in-vivo:in-vitro ratios than ggTas2r-selective ligands. In-vivo thresholds were similar or up to two orders of magnitude higher than the in-vitro ones.

Toward the rational design of macrolide antibiotics to combat resistance.

Macrolides, one of the most prescribed classes of antibiotics, bind in the bacterial ribosome's polypeptide exit tunnel and inhibit translation. However, mutations and other ribosomal modifications, especially to the base A2058 of the 23S rRNA, have led to a growing resistance problem. Here, we have used molecular dynamics simulations to study the macrolides erythromycin and azithromycin in wild-type, A2058G-mutated, and singly or doubly A2058-methylated Escherichia coli ribosomes. We find that the ribosomal modifications result in less favorable interactions between the base 2058 and the desosamine sugar of the macrolides, as well as greater displacement of the macrolides from their crystal structure position, illuminating the causes of resistance. We have also examined four azithromycin derivatives containing aromatic indole-analog moieties, which were previously designed based on simulations of the stalling peptide SecM in the ribosome. Surprisingly, we found that the studied moieties could adopt very different geometries when interacting with a key base in the tunnel, A751, possibly explaining their distinct activities. Based on our simulations, we propose modifications to the indole-analog moieties that should increase their interactions with A751 and, consequently, enhance the potency of future azithromycin derivatives.

Development of Novel Erythromycin Derivatives with Inhibitory Activity against Proliferation of Tumor Cells.

In our continuing structure-activity relationship study of a new class of erythromycin A (EM-A) derivatives with antiproliferative activity, a new series of de(N-methyl) EM-A dimers jointed by a four-atom linker, -CH2CH = CHCH2-, were prepared and their antiproliferative activity against three human tumor cell lines was evaluated by MTT assay. The most active EM-A dimer, compound 1b, that carrying C6 methoxyl groups was further investigated and showed potent antiproliferative activity in six other human tumor cell lines. Flow cytometry analysis of 1b treated HeLa and MCF-7 cells indicated that the four-atom EM-A dimers induced the SubG1 phase cell cycle arrest and cell apoptosis, in time- and dose-dependent manners. Further experiments including morphologic observation, DNA agarose gel electrophoresis, mitochondrial potential alternation and western blot analysis revealed that the antiproliferative mechanism may involve the induction of apoptosis in activating the mitochondrial pathway, and regulation of apoptotic proteins.

Removal of Penicillin G and Erythromycin with Ionizing Radiation Followed by Biological Treatment.

The decomposition of penicillin G and erythromycin antibiotics at concentration of 0.2 mg ml(-1) by gamma irradiation at 50 kGy followed by biological treatment with Cupriavidus metallidurans CH34 was evaluated. Degradation of penicillin G and erythromycin was analyzed using nuclear magnetic resonance analysis (NMR), fourier transform infrared spectroscopy (FTIR), and chemical oxygen demand (COD). The exposure to the absorbed dose of 50 kGy caused degradation of penicillin G and erythromycin in the aqueous solution. The complete disappearance of NMR and FTIR peaks following irradiation confirmed the breakage of the ß-lactam ring in penicillin G, and the decarboxylation and cleavage of the thiazolidine ring and for erythromycin, the complete destruction of the three aromatic rings. Irradiation alone removed 52.8 and 65.5 % of penicillin G and erythromycin, respectively. Further reduction to 12.6 and 14 % of the original penicillin G and erythromycin COD, respectively, was achieved using treatment of the irradiation products with C. metallidurans.

A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest.

Nascent polypeptides can induce ribosome stalling, regulating downstream genes. Stalling of ErmBL peptide translation in the presence of the macrolide antibiotic erythromycin leads to resistance in Streptococcus sanguis. To reveal this stalling mechanism we obtained 3.6-Å-resolution cryo-EM structures of ErmBL-stalled ribosomes with erythromycin. The nascent peptide adopts an unusual conformation with the C-terminal Asp10 side chain in a previously unseen rotated position. Together with molecular dynamics simulations, the structures indicate that peptide-bond formation is inhibited by displacement of the peptidyl-tRNA A76 ribose from its canonical position, and by non-productive interactions of the A-tRNA Lys11 side chain with the A-site crevice. These two effects combine to perturb peptide-bond formation by increasing the distance between the attacking Lys11 amine and the Asp10 carbonyl carbon. The interplay between drug, peptide and ribosome uncovered here also provides insight into the fundamental mechanism of peptide-bond formation.

Enhancement of the properties of a drug by mono-deuteriation: reduction of acid-catalysed formation of a gut-motilide enol ether from 8-deuterio-erythromycin B.

Erythromycin B is structurally very similar to erythromycin A, and also shares its clinically important antibacterial activity. Its potential advantage is that it is much more stable to acid. Both compounds are susceptible to 6-9-enol ether formation, involving loss of a proton from C-8. The enol ethers lack antibacterial activity and can give rise to unpleasant gut motilide side-effects. Our previous work on degradation kinetics revealed that the formation of erythromycin B enol ether from erythromycin B is subject to a large deuterium isotope effect. We therefore synthesized 8-d-erythromycin B (in 87% yield) in the hope that acid-catalysed enol ether formation would be reduced, relative to erythromycin B. In a range of microbiological and biochemical assays, deuteriation did not appear to compromise the efficacy of the drug. Degradation studies showed, however, that incorporation of deuterium into erythromycin B reduces (though does not completely suppress) enol ether formation, providing the possibility of using a facile mono-deuteriation to reduce the gut motilide side-effects of the drug.

Non-antibiotic 12-membered macrolides: design, synthesis and biological evaluation in a cigarette-smoking model.

The 14-membered macrolide erythromycin A expresses three distinct biological properties, including antibacterial activity, gastrointestinal motor-stimulating activity and anti-inflammatory and/or immunomodulatory effects. Although low-dose, long-term therapy using 14- and 15-membered macrolides displaying anti-inflammatory and/or immunomodulatory activity effectively treats diffuse panbronchiolitis and chronic sinusitis, bacterial resistance may emerge. To address this issue, we developed the 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900) that promotes monocyte to macrophage differentiation, a marker for anti-inflammatory and/or immunomodulatory effects, without possessing antibacterial activity. In this article, we report that the new macrolide derivative (8R,9S) -de(3'-N-methyl)-3'-N-(p-chlorobenzyl)-de(3-O-cladinosyl)-3-dehydro-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A 12,13-carbonate (EM939) exhibited stronger promotive activity for monocyte to macrophage differentiation than that of the parent compound EM900 in addition to reduced cytotoxicity toward THP-1 cells and antibacterial inactivity. In a cigarette-smoking model used to simulate chronic obstructive pulmonary disease (COPD), the EM900 derivatives significantly attenuated lung and alveolar inflations, functionally and histologically, via oral administration. Because of these marked therapeutic effects, non-antibiotic EM900 derivatives may become central to the treatment of chronic inflammatory diseases such as COPD.

Site-Selective Reactions with Peptide-Based Catalysts.

The problem of catalyst-controlled site-selectivity can potentially require a catalyst to overcome energetic barriers larger than those associated with enantioselective reactions. This challenge is a signature of substrates that present reactive sites that are not of equivalent reactivity. Herein we present a narrative of our laboratory's efforts to overcome this challenge using peptide-based catalysts. We highlight the interplay between understanding the inherent reactivity preferences of a given target molecule and the development of catalysts that can overcome intrinsic preferences embedded within a substrate.

Effects of Mentha suaveolens essential oil on Chlamydia trachomatis.

Chlamydia trachomatis, the most common cause of sexually transmitted bacterial infection worldwide, has a unique biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body. C. trachomatis is responsible for severe reproductive complications including pelvic inflammatory disease, ectopic pregnancy, and obstructive infertility. The aim of our study was to evaluate whether Mentha suaveolens essential oil (EOMS) can be considered as a promising candidate for preventing C. trachomatis infection. Specifically, we investigated the in vitro effects of EOMS towards C. trachomatis analysing the different phases of chlamydial developmental cycle. Our results demonstrated that EOMS was effective towards C. trachomatis, whereby it not only inactivated infectious elementary bodies but also inhibited chlamydial replication. Our study also revealed the effectiveness of EOMS, in combination with erythromycin, towards C. trachomatis with a substantial reduction in the minimum effect dose of antibiotic. In conclusion, EOMS treatment may represent a preventative strategy since it may reduce C. trachomatis transmission in the population and, thereby, reduce the number of new chlamydial infections and risk of developing of severe sequelae.

Drug sensing by the ribosome induces translational arrest via active site perturbation.

During protein synthesis, nascent polypeptide chains within the ribosomal tunnel can act in cis to induce ribosome stalling and regulate expression of downstream genes. The Staphylococcus aureus ErmCL leader peptide induces stalling in the presence of clinically important macrolide antibiotics, such as erythromycin, leading to the induction of the downstream macrolide resistance methyltransferase ErmC. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmCL-stalled ribosome at 3.9 Å resolution. The structure reveals how the ErmCL nascent chain directly senses the presence of the tunnel-bound drug and thereby induces allosteric conformational rearrangements at the peptidyltransferase center (PTC) of the ribosome. ErmCL-induced perturbations of the PTC prevent stable binding and accommodation of the aminoacyl-tRNA at the A-site, leading to inhibition of peptide bond formation and translation arrest.

Effect of erythromycin-doped calcium polyphosphate scaffold composite in a mouse pouch infection model.

We previously showed that strontium-doped calcium polyphosphate (SCPP) scaffold with poly(vinyl alcohol) (PVA) coating extended the impregnated erythromycin (EM) release. In this study, we examined the bactericidal effect of EM-doped SCPP (SCPP(EM) ) scaffolds with PVA coating in a Staphylococcus aureus (S. aureus) infected mouse pouch. SCPP scaffolds with or without 5% EM, and SCPP(EM) scaffolds coated with PVA (with or without 5% EM) were prepared. Scaffolds were implanted in the pouch of BALB/c mice, followed by inoculation of 1 × 10(3) colony-forming units of S. aureus. Mice were sacrificed 14 days after surgery. Pouch tissues and scaffolds were collected for histology, scanning electron microscopy, and microbiological analysis. In the absence of SCPP scaffolds, the pouch infection was eliminated by the host immune surveillance. In the presence of SCPP scaffolds, both the pouch tissues and scaffolds were infected, but SCPP(EM) scaffolds successfully inhibited bacterial growth. Although PVA coating of SCPP(EM) scaffolds enhanced bacterial growth, incorporation of EM into PVA coating inhibited growth. In conclusion, BALB/c mice were capable of eradicating a low grade S. aureus infection. SCPP protected S. aureus growth from host immune surveillance. Though PVA coating sustained EM release in vitro, it was unable to inhibit bacterial growth because PVA gel matrix provided a temporary shelter for bacteria to grow and slowed the EM release from SCPP scaffold. To guarantee a sufficient inhibition of bacterial growth at the initial stage, embedding EM or other antibiotics in the PVA coating is also essential.