Erythromycin Derivatives Research Articles

Potentials of Polymeric Nanocarriers Loaded with Clarithromycin for Antibacterial Activity

Clarithromycin (CTM) is a semisynthetic derivative of erythromycin that have been reported to exhibit potential antibiotic activities mostly against the gram-negative and gram-positive bacteria, lower/upper respiratory tract and skin infection causing pathogens. It is widely used for the prevention and management of infections due to Mycobacterium avium complexes and peptic ulcers due to Helicobacter pylori. Various marketed formulation of CTM in the form of tablets, capsules and other conventional dosage forms is available as anti-infective, however the drug itself has several limitations. These limitations include low oral bioavailability, rapid metabolism, poor targeting to infected sites and toxicity to normal cells/tissues. Also, the CTM-based therapy has been reported for several gastrointestinal adverse effects, including diarrhea, stomach upset, gastric distress, atypical taste, and others. Thus, to overcome these issue, various novel strategies including nanotechnology or nanocarrier-based approaches have showed significant effects and have been immensely considered worldwide. In recent years, the various nanocarriers or nanocarrier-based delivery systems, particularly the polymeric nanocarrier have played significant role in effective drug targeting. Thus, in this review, the various polymeric nanocarrier-based delivery systems of CTM that effective reduced the dosing frequency, improved the patient compliance and potentially enhanced the therapeutic efficiency of CTM has been summarized.

PhoP- and GlnR-mediated regulation of metK transcription and its impact upon S-adenosyl-methionine biosynthesis in Saccharopolyspora erythraea

BackgroundErythromycin A (Er A) has a broad antibacterial effect and is a source of erythromycin derivatives. Methylation of erythromycin C (Er C), catalyzed by S-adenosyl-methionine (SAM)-dependent O-methyltransferase EryG, is the key final step in Er A biosynthesis. Er A biosynthesis, including EryG production, is regulated by the phosphate response factor PhoP and the nitrogen response factor GlnR. However, the regulatory effect of these proteins upon S-adenosyl-methionine synthetase (MetK) production is unknown.ResultsIn this study, we used bioinformatics approaches to identify metK (SACE_3900), which codes for S-adenosyl-methionine synthetase (MetK). Electrophoretic mobility shift assays (EMSAs) revealed that PhoP and GlnR directly interact with the promoter of metK, and quantitative PCR (RT-qPCR) confirmed that each protein positively regulated metK transcription. Moreover, intracellular SAM was increased upon overexpression of either phoP or glnR under phosphate or nitrogen limited conditions, respectively. Finally, both the production of Er A and the transformation ratio from Er C to Er A increased upon phoP overexpression, but surprisingly, not upon glnR overexpression.ConclusionsManipulating the phosphate and nitrogen response factors, PhoP and GlnR provides a novel strategy for increasing the yield of SAM and the production of Er A in Saccharopolyspora erythraea .

Selected Derivatives of Erythromycin B-In Silico and Anti-Malarial Studies.

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 of 68.6 µM, d-erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum. Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P. falciparum is the reason for their weak in vitro anti-malarial activities.

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.

Focus on azithromycin

Azithromycin is a second-generation macrolide that was developed in the early 1980s as a semi-synthetic derivative of erythromycin. This broad-spectrum antibiotic is known for its activity against some Gram-negative organisms such as H. influenza. Azithromycin is similar to other macrolides, however, it also has the ability to inhibit quorum sensing and the formation of biofilm. Azithromycin has been beneficial in the treatment of influenza and Middle East respiratory syndrome coronavirus (MERS-CoV) and recently has shown to be effective against severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) when used in combination with hydroxychloroquine or chloroquine. The side-effect that needs to be monitored carefully is the risk of cardiotoxicity when azithromycin is combined with chloroquine or hydroxychloroquine.

Azithromycin – more than an antibiotic

Azithromycin, which belongs to the azalide subclass, is a derivative of erythromycin, obtained by inserting a methyl substituted nitrogen atom into the lactone ring. In this way, better stability at the low pH of the gastric juice, improved bioavailability, and an extended half-life relative to erythromycin were achieved. The mechanism of azithromycin antibacterial activity is to inhibit bacterial cell protein synthesis by binding to the 50S ribosomal subunit and preventing bacterial mRNA translation and protein biosynthesis. In addition to bactericidal activity, azithromycin also has immunomodulatory, anti-inflammatory, and antiviral properties. For this reason, it is used in exacerbations of asthma and chronic obstructive pulmonary disease. However, its antibacterial efficacy and high safety are most important. In clinical practice, several model clinical profiles of patients can be presented: children with community-acquired Mycoplasma pneumoniae pneumonia, recurrent respiratory tract infections and exacerbations of chronic inflammations, acute bronchitis of likely bacterial aetiology, tonsillitis and allergy to β-lactam antibiotics, and pertussis. Efficacy has also been proven in primary skin and soft tissue infections as well as erysipelas. However, azithromycin may also have an unusual use, as confirmed by recent reports. These include travellers’ diarrhoea, cat scratch disease (bartonellosis – rickettsiosis), and mycobacteriosis (nontuberculous mycobacteria). Appropriate spectrum of action, efficacy and safety, as well as a low percentage of resistant bacteria, good bioavailability, easy to remember, practical and simple dosage regimen and optimal cost are features of a good antibiotic. Azithromycin is close to meeting all these criteria.

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.

LC-MS/MS profile of an active pharmaceutical ingredient and its impurities in commercial preparation

The azithromycin is a semisynthetic derivative of erythromycin A, and commercially produced by a series of chemical reaction involved Beckmann rearrangement followed by reductive methylation. Several azithromycin related compounds (ARCs) have been identified as impurity in the commercial products and the processes. These ARCs probably generated from impure starting material; a mixture of different erythromycin. Literature search revealed that approximately 90 erythromycins had been isolated and characterized so far. In current study, we have identified many new ARCs, including previously reported by detailed LC-MS/MS studies. In this study, first, the structures of ARCs were assumed by probable chemical transformation method; erythromycin A to azithromycin A; and, second, assumed structures of ARCs were confirmed by HRMS within the acceptable ppm error limit (±5 ppm) followed by MS/MS fragmentation to identify the characteristic common ion fragments and by precursor ion scan.

The Research of General Reaction: Synthesis of Erythromycin Derivatives Impurities about N-demonomethyl Compounds

Erythromycin derivatives have been used as antibacterial drugs in clinic for many years. As lacking of relevant impurities, those drugs quality control are limited. So, we carried out a research of general reaction: synthesis of 3 “- N-demonomethyl - Erythromycin, 3 “- N-demonomethyl - Clarithromycin, 3 “- N- demonomethyl - Roxithromycin and 3 “- N-demonomethyl- Azithromycin by using Erythromycin, Clarithromycin, Roxithromycin and Azithromycin as the raw material respectively. Using 1HNMR, 13CNMR and MS identified those impurities structures. Our methods have many advantages: raw materials cheap, getting easily, safely and stability, high yield and good purity, and it also can use for quality control of Erythromycin derivatives and new drug development based on 3“-N-demonomenthyl macrolide compounds.

24 エリスロマイシン誘導体のO-メチル化における位置選択性について(口頭発表の部)

24 エリスロマイシン誘導体のO-メチル化における位置選択性について(口頭発表の部)

Design, synthesis and structure-bactericidal activity relationships of novel 9-oxime ketolides and reductive epimers of acylides

Erythromycin was long viewed as a bacteriostatic agent. The erythromycin derivatives, 9-oxime ketolides have a species-specific bactericidal profile. Among them, the 3′-allyl version of the 9-oxime ketolide 1 (Ar=3-quinolyl; 17a) is bactericidal against Streptococcus pneumoniae and Streptococcus pyogenes. In contrast, the 2-fluoro analogs of 1, 13a (Ar=6-quinolyl), 13b (Ar=3-quinolyl) and 24a (Ar=4-isoquinolyl), show bactericidal activities against S. pneumoniae, Staphylococcus aureus and Moraxella catarrhalis, while the 2-fluoro analogs 13c (Ar=3-aminopyridyl) and 24b (Ar=3-carbamoylpyridyl) are only bactericidal against S. pneumoniae and Haemophilus influenzae. Reduction of the ketolides led to novel epiacylides, the 3-O-epimers of the acylides. Alteration of linker length (30b vs. 30a), 2-fluorination (33 vs. 30a) and incorporation of additional spacers at the 9-oxime or 6-OH (35, 40 vs. 30a) did not restore the epiacylides back to be as active as the acylide 31. Molecular docking suggested that epimerization at the 3-position reshapes the orientation of the 3-O-sidechain and leads to considerably weaker binding with bacterial ribosomes.

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.

Mechanism of synergy between SIPI-8294 and β-lactam antibiotics against methicillin-resistant Staphylococcus aureus.

SIPI-8294, as an erythromycin derivative, has only weak antibacterial effects on MRSA and MSSA. Interestingly, synergistic effect of SIPI-8294 with oxacillin was observed both invitro and invivo. Western blot and RT-PCR results demonstrate that mecA expressions were suppressed by SIPI-8294 in MRSA. Furthermore, the knock out of mecA in ATCC 43300 led to the loss of synergy of the combinations while mecA complemented strain showed almost the same synergistic capability compared to the wild type strain. However, the knock out of mecR1 and mecI in MRSA displayed no impact on the synergy of the combinations and the ability of SIPI-8294 to suppress mecA expression. In summary, our study has demonstrated that SIPI-8294 could dramatically reverse MRSA resistance to β-lactams both invitro and invivo owing to inhibiting mecA expression. However, mecR1 and mecI, as the pivotal regulatory genes of mecA, do not participate in SIPI-8294-mecA pathway. The research indicates that it may be a promising strategy for combating MRSA infections with the combinations of SIPI-8294 and β-lactam antibiotics. The research of the mechanism is important for structure modification and new drug development. This study is the first report on the mechanism of synergy between SIPI-8294 and β-lactams against MRSA on the molecular level. In this study, SIPI-8294 showed strong synergistic effects on β-lactam antibiotics both invitro and invivo owing to inhibiting mecA expression. As pivotal regulatory genes of mecA, mecR1 and mecI do not participate in SIPI-8294-mecA pathway and are not involved in the synergism of SIPI-8294 and β-lactams. The research indicates that it may be a promising strategy for combating MRSA infections with the combinations of SIPI-8294 and β-lactams. The research is important for structure modification and new drug development.

Mechanisms of the Macrolide-Induced Inhibition of Superoxide Generation by Neutrophils.

The effect of macrolides on the superoxide (O2 (-)) production by neutrophils was studied. Resting neutrophils become primed by lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), and primed neutrophils generate O2 (-) in response to fMLP or adhesion, respectively. Both LPS-primed fMLP-stimulated O2 (-) generation by macrolide-treated neutrophils and adhesion-stimulated O2 (-) generation by macrolide-treated fMLP-primed neutrophils were inhibited. Macrolide inhibition of O2 (-) generation was dependent on serum or pH. Serum could be substituted by NaHCO3. The intensity of inhibition was azithromycin = roxithromycin > clarithromycin > erythromycin, in that order. Non-antimicrobial derivatives of erythromycin, that is, EM703 and EM900, inhibited O2 (-) generation at pH 7.4. NH4Cl abolished the activity of azithromycin (AZ) only when added to neutrophils with AZ but not after incubation with AZ, suggesting that NH4Cl prevented the influx of AZ. AZ did not affect the expression of alkaline phosphatase, CD11b, and cytochrome b558 in both resting and LPS-primed neutrophils. These results suggested that macrolides did not affect granule mobilization but inhibited O2 (-) generation selectively.

Proteomic response of methicillin-resistant S. aureus to a synergistic antibacterial drug combination: a novel erythromycin derivative and oxacillin.

The use of antibacterial drug combinations with synergistic effects is increasingly seen as a critical strategy to combat multi-drug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). In this work, the proteome responses in MRSA under the stress of a sub-inhibitory dose of a synergistic drug combination of a novel erythromycin derivative, SIPI-8294, and oxacillin, were studied by label-free quantitative proteomics. Several control treatment groups were designed to isolate proteome responses potentially related to the synergy: (1) the non-synergistic drug combination of erythromycin and oxacillin, (2) SIPI-8294 only, (3) oxacillin only and (4) erythromycin only. Results showed that 200 proteins were differentially expressed in SIPI-8294/oxacillin-treated cells. Among these proteins, the level of penicillin binding protein 2a, the protein mainly responsible for oxacillin resistance in MRSA, was four times lower in the SIPI-8294/oxacillin group than in the erythromycin/oxacillin group, suggesting that SIPI-8294 may interfere with this known oxacillin resistance mechanism. Moreover, hierarchical clustering analysis of differentially expressed proteins under different treatments revealed that SIPI-8294/oxacillin elicits very different responses than the individual drugs or the non-synergistic erythromycin/oxacillin combination. Bioinformatic analysis indicated that the synergistic effect can be further traced to a disruption in oxidation-reduction homeostasis and cell wall biosynthesis.

Immune Modulation in Children. A South African Perspective

Immune deficiencies in South Africa can be divided into two categories, i.e. primary immune deficiencies or secondary due to illnesses such as malignancies or infection e.g. the human immune virus (HIV). In the private pediatric clinical setting, children of-ten present with illnesses that can be traced to selective secondary immune deficiencies. Although highly effective, not all children are eligible for intravenous immunoglobulin therapy, either due to costs or non-IgG related immune deficiencies such as selective IgA or T-cell deficiencies. Intramuscular and subdermal immunoglobulin options are equally expensive and uncomfortable. The use of oral immune modulators, such as the erythromycin derivatives, has raised some concern, mainly because of the potential risk of bacterial resistance. Other so called immune boosters, e.g. Echinacea’s, are being advertised as potential solutions but have yet to prove to be efficient. In addition to this, the interaction between the gut micro-biome and the immune system is complex, but critical in maintaining and optimizing the immune system. Under certain disease entities such as inflammatory bowel disease, resulting in a leaky gut, the immune system becomes compromised because of the interaction between certain inflammatory signals and the lymphoid and bone marrow tissue specifically the naive and memory T-cell populations, such as CD4 and CD8 lymphocytes. An overview to immune modulation will be presented that has a direct beneficial effect on the health outcome of the children and new therapeutic modalities will be evaluated in terms of their response and restoration of the T-cell populations.

Recovery and separation of erythromycin from industrial wastewater by imprinted magnetic nanoparticles that exploit β-cyclodextrin as the functional monomer.

A type of surface imprinting over magnetic Fe3 O4 nanoparticles utilizing erythromycin-A as a template for use in the separation and recovery of erythromycin was developed and investigated. As the intermolecular forces play a key role in the performance of imprinted materials, differential scanning calorimetry, and (1) H NMR spectroscopy was employed to evaluate the interactions between erythromycin and the functional monomer β-cyclodextrin. To synthesize the surface imprinted polymers, magnetic Fe3 O4 nanoparticles, the core materials, were modified with a free radical initiator to initialize polymerization in a "grafting from" manner. Then using acryloyl-modified β-cyclodextrin as the functional monomer and ethyleneglycol dimethacrylate as the cross-linker, thin erythromycin-imprinted films were fabricated by the radical-induced graft copolymerization of monomers on the surface of the Fe3 O4 nanoparticles. Selectivity experiments showed that the erythromycin-A-imprinted materials had recognition ability toward erythromycin derivatives. Finally, these magnetic molecularly imprinted particles were successfully used for the separation and enrichment of erythromycin from the mother liquor. The recovery, detected by high-performance liquid chromatography and differential pulse voltammetry, approached 97%. The combination of the specific selectivity of the imprinted material and the magnetic separation provided a powerful tool that is simple, flexible, and selective for the separation and recovery of erythromycin.

Modeling Interactions of Erythromycin Derivatives with Ribosomes.

Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3'-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3-oxopropyl group in the 3'-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.

Substrate Flexibility of a Mutated Acyltransferase Domain and Implications for Polyketide Biosynthesis

Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzyme's substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification.

Enantioseparation of basic chiral drugs on a carbamoylated erythromycin-zirconia hybrid monolith using capillary electrochromatography

An organic–inorganic hybrid monolithic column was prepared within the confines of a capillary via a single-step in situ sol–gel approach using zirconium tetrabutoxide as a precursor to compose the inorganic backbone and 3-triethoxysilylpropyl carbamoylated derivative of erythromycin (TEOSPC-ERY) as a co-precursor to introduce the organic chiral selector moiety in the zirconia backbone. The resulting carbamoylated ERY-zirconia hybrid monolith (ERY-ZHM) showed homogeneous morphology with well-defined through pores and was tightly connected with the inner wall of the capillary. The column was employed for capillary electrochromatographic enantioseparation of six basic chiral drugs in mobile phases (MPs) consisting of acetonitrile (ACN) and triethylammonium acetate (TEAA) buffer. The effects of composition of MP and applied voltage on chiral separation were investigated by using propranolol as a representative analyte. The highest resolution (Rs=3.33) was obtained with a MP consisting of 10/90 (v/v) ACN/TEAA buffer (10mM, pH 7), 10kV applied voltage and 25°C capillary temperature. The relative standard deviations for resolution values regarding run to run, day to day, column to column and batch to batch repeatability were 0.41%, 0.89%, 1.80% and 2.26% (for n=3), respectively, indicating satisfactory stability of columns and reproducibility of column preparation process.