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Journal of the American Society For... Aug 2019The 14- and 16-membered macrolide antibiotics are an important structural class. Ubiquitously produced by a number of bacterial strains, namely actinomycetes,...
The 14- and 16-membered macrolide antibiotics are an important structural class. Ubiquitously produced by a number of bacterial strains, namely actinomycetes, purification and structure elucidation of the wide array of analogs is challenging, both for discovery efforts and methodologies to monitor for byproducts, metabolites, and contaminants. Collision-induced dissociation mass spectrometry offers an attractive solution, enabling characterization of mixtures, and providing a wealth of structural information. However, interpretation of these spectra can be difficult. We present a study of 14- and 16-membered macrolide antibiotics, including MS analysis for unprecedented depth of coverage, and complimentary analysis with DO and HO labeling to elucidate fragmentation mechanisms. These analyses contrast the behaviors of varying classes of macrolides and highlight how analogues can be identified in relation to similar structures, which will provide utility for future studies of novel macrolides, as well as impurities, metabolites, and degradation products of pharmaceuticals. Graphical Abstract.
Topics: Anti-Bacterial Agents; Deuterium; Erythromycin; Josamycin; Leucomycins; Macrolides; Oleandomycin; Spectrometry, Mass, Electrospray Ionization; Spiramycin; Tylosin; Water
PubMed: 30993640
DOI: 10.1007/s13361-019-02210-w -
Journal of Pharmaceutical and... Sep 2020A lateral flow immunoassay (LFIA) using latex particles labeled with antibody to BSA-clarithromycin (CLA) was developed for the rapid simultaneous group determination of...
A lateral flow immunoassay (LFIA) using latex particles labeled with antibody to BSA-clarithromycin (CLA) was developed for the rapid simultaneous group determination of six macrolide antibiotics. Optimization of antigen spotting on the membrane and latex probe loading allowed improving visual detectability (vLOD) 100 times, which was 1, 1, 10, 10, 50, and 1000 ng/mL for CLA, roxithromycin, erythromycin, dirithromycin, azithromycin, and oleandomycin in buffer, respectively. The calculated limits of instrumental detection (cLOD) were respectively 0.12, 0.15, 1.4, 2.1, 2.4, and 3.3 ng/mL. To avoid a strong influence of breast milk of a very diverse and variable composition, a sample pretreatment is proposed. The six macrolides mentioned can be visually detected in breast milk after 20 min pretreatment at concentrations of 10-1000 ng/mL or instrumentally with cLOD of 4.0, 2.5, 30, 42, 42 and 180 ng/mL. The recovery rate from the spiked samples carried out using a strip scanner device ranged from 71 % to 110 %, and precision expressed as relative standard deviation was between 3-14 %. The described rapid on-site diagnostic assay format can be useful for monitoring the content of antibiotics in breast milk during macrolide treatment to ensure safe breastfeeding of infants.
Topics: Anti-Bacterial Agents; Clarithromycin; Humans; Immunoassay; Macrolides; Microspheres; Milk, Human
PubMed: 32693204
DOI: 10.1016/j.jpba.2020.113450 -
Drug Metabolism and Disposition: the... Oct 2023The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam...
The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 M in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 M) and CBZ (100 or 1000 M) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction.
Topics: Humans; Cytochrome P-450 CYP3A; Midazolam; Troleandomycin; Clarithromycin; Microsomes, Liver; Drug Interactions; Carbamazepine
PubMed: 37524542
DOI: 10.1124/dmd.123.001382 -
Biomolecules Oct 2020The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated...
The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein-substrate interactions. docking of the monoglycosylated L-O-DEO in the closed OleP-DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.
Topics: Catalysis; Crystallography, X-Ray; Cytochrome P-450 Enzyme System; Lactones; Protein Domains; Rhamnose; Structure-Activity Relationship; Substrate Specificity
PubMed: 33036250
DOI: 10.3390/biom10101411 -
PloS One 2023Lactic acid bacteria are known to produce numerous antibacterial metabolites that are active against various pathogenic microbes. In this study, bioactive metabolites...
Lactic acid bacteria are known to produce numerous antibacterial metabolites that are active against various pathogenic microbes. In this study, bioactive metabolites from the cell free supernatant of Loigolactobacillus coryniformis BCH-4 were obtained by liquid-liquid extraction, using ethyl acetate, followed by fractionation, using silica gel column chromatography. The collected F23 fraction effectively inhibited the growth of pathogenic bacteria (Escherichia coli, Bacillus cereus, and Staphylococcus aureus) by observing the minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC). The evaluated values of MIC were 15.6 ± 0.34, 3.9 ± 0.59, and 31.2 ± 0.67 μg/mL and MBC were 15.6 ± 0.98, 7.8 ± 0.45, and 62.5 ± 0.23 μg/mL respectively, against the above-mentioned pathogenic bacteria. The concentration of F23 fraction was varying from 1000 to 1.9 μg/mL. Furthermore, the fraction also exhibited sustainable biofilm inhibition. Using the Electrospray Ionization Mass Spectrometry (ESI-MS/MS), the metabolites present in the bioactive fraction (F23), were identified as phthalic acid, myristic acid, mangiferin, 16-hydroxylpalmatic acid, apigenin, and oleandomycin. By using in silico approach, docking analysis showed good interaction of identified metabolites and receptor proteins of pathogenic bacteria. The present study suggested Loigolactobacillus coryniformis BCH-4, as a promising source of natural bioactive metabolites which may receive great benefit as potential sources of drugs in the pharmacological sector.
Topics: Humans; Tandem Mass Spectrometry; Anti-Bacterial Agents; Staphylococcus aureus; Bacillus cereus; Microbial Sensitivity Tests
PubMed: 37561679
DOI: 10.1371/journal.pone.0289723 -
Huan Jing Ke Xue= Huanjing Kexue Sep 2023Antibiotic contamination in drinking water has attracted widespread attention. The pollution condition of six macrolide antibiotics (erythromycin-H[KG-*2/5]O,...
Antibiotic contamination in drinking water has attracted widespread attention. The pollution condition of six macrolide antibiotics (erythromycin-H[KG-*2/5]O, clarithromycin, oleandomycin, roxithromycin, leucomycin, and tylosin) in two drinking water treatment plants was monitored, and the reaction mechanism of tylosin, a typical macrolide antibiotic, during chlorination disinfection treatment was investigated. The results showed that the six macrolide antibiotics can be widely detected in the drinking water treatment processes; however, their concentrations were generally very low. The concentrations of macrolide antibiotics in the influents and effluents ranged from 0.18 ng·L to 3.97 ng·L and 0.02 ng·L to 1.91 ng·L, respectively. The removal rates of the six macrolides in the drinking water treatment were different, ranging from 18% (oleandomycin) to 100% (erythromycin- H[KG-*2/5]O). The degradation of the six macrolides during chlorination was slow and greatly affected by water quality parameters. The chlorination degradation of tylosin followed the second-order reaction kinetic mode, with the kinetic rate constant of 0.77 L·(mol·s) at pH 7.0. Nine chlorination degradation products of tylosin were detected, and the reaction pathways primarily included tertiary amine hydroxylation, aromatic oxidation, and epoxy addition.
Topics: Tylosin; Drinking Water; Halogenation; Anti-Bacterial Agents; Macrolides; Erythromycin; Oleandomycin
PubMed: 37699819
DOI: 10.13227/j.hjkx.202209221 -
Journal of Environmental Science and... 2021A simple, rapid and sensitive screening method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the identification of 7 macrolides...
A simple, rapid and sensitive screening method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the identification of 7 macrolides (clarithromycin, erythromycin, oleandomycin, spiramycin, tilmicosin, troleandomycin and tylosin) and 8 quinolones (ciprofloxacin, difloxacin, enrofloxacin, flumequine, moxifloxacin, nalidixic acid, norfloxacin and ofloxacin) in meat and egg-based baby foods. Sample preparation was performed using an alkaline modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction method without additional clean-up steps. A simplex-lattice mixture experimental design was used in the optimization of the QuEChERS extraction solvent. The developed method was successfully validated according to the Commission Decision 2002/657/EC and the European Community Reference Laboratories Residues Guidelines regarding the validation of screening methods 20/01/2010, adopting a fixed permited tolerance for relative ion ratio. Samples of baby food (n = 44) commercialized in Rio de Janeiro, Brazil, were analyzed using the validated method and none of them presented residues of the searched macrolides and quinolones, with a screening target value of 5 µg kg.
Topics: Animals; Anti-Bacterial Agents; Chemical Fractionation; Chromatography, Liquid; Drug Residues; Eggs; Food Analysis; Food Contamination; Infant Food; Macrolides; Meat; Quinolones; Tandem Mass Spectrometry
PubMed: 33463404
DOI: 10.1080/03601234.2021.1872324 -
Diagnostic Microbiology and Infectious... May 2024Haemophilus influenzae is an important pathogen able to cause various forms of respiratory and invasive disease. To provide high sensitivity for detection, culture media...
Haemophilus influenzae is an important pathogen able to cause various forms of respiratory and invasive disease. To provide high sensitivity for detection, culture media must inhibit growth of residential flora from the respiratory tract. This study aimed to identify and compare the diagnostic and economic advantages of using bacitracin containing selective agar (SEL) or oleandomycin disk supplemented chocolate agar (CHOC). Growth and semi-quantitative abundance of H. influenzae and growth suppression of residential flora was prospectively assessed in a 28-week period. H. influenzae was identified in 164 (5 %) of all included samples: CHOC and SEL, CHOC only, and SEL only were positive in 95, 24, and 45 cases. Diagnostic superiority of SEL was primarily attributable to the results of throat swabs. However, on average, € 200 had to be spent for the detection of each additional isolate that was recovered only because of additional incubation on SEL.
Topics: Humans; Agar; Bacitracin; Haemophilus influenzae; Oleandomycin; Chocolate; Culture Media
PubMed: 38422664
DOI: 10.1016/j.diagmicrobio.2024.116203 -
Biotechnology Letters May 2020Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of...
OBJECTIVE
Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus.
RESULTS
Co-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6β-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L h. NMR spectroscopy confirmed the identity of MDCA as the sole product.
CONCLUSIONS
The multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.
Topics: Bacterial Proteins; Biocatalysis; Cloning, Molecular; Cytochrome P-450 Enzyme System; Deoxycholic Acid; Escherichia coli; Hydroxylation; Lithocholic Acid; Oxidoreductases; Pseudomonas putida; Streptomyces antibioticus
PubMed: 31974648
DOI: 10.1007/s10529-020-02813-4 -
Foods (Basel, Switzerland) Mar 2024The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin...
Dispersive Solid-Phase Extraction and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry-A Rapid and Accurate Method for Detecting 10 Macrolide Residues in Aquatic Products.
The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin (KIT), josamycin (JOS), spiramycin (SPI), tilmicosin (TIL), tylosin (TYL), and roxithromycin (ROX), was determined using solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The residues were extracted with 1% ammonia acetonitrile solution and purified by neutral alumina adsorption. Chromatographic separation was completed on an ACQUITY UPLC BEH C column with acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and mass spectrometry detection was performed by multiple reaction monitoring scanning with the positive mode in an electrospray ion source (ESI). Five isotopically labeled compounds were used as internal standards for quality control purposes. The findings indicated that across the mass concentration span of 1.0-100 μg/L, there was a strong linear correlation ( > 0.99) between the concentration and instrumental response for the 10 MALs. The limit of detection of UPLC-MS/MS was 0.25-0.50 μg/kg, and the limit of quantitation was 0.5-1.0 μg/kg. The added recovery of blank matrix samples at standard gradient levels (1.0, 5.0, and 50.0 μg/kg) was 83.1-116.6%, and the intra-day precision and inter-day precisions were 3.7 and 13.8%, respectively. The method is simple and fast, with high accuracy and good repeatability, in line with the requirements for accurate qualitative and quantitative analysis of the residues for 10 MALs in aquatic products.
PubMed: 38540855
DOI: 10.3390/foods13060866