-
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 -
Enzyme and Microbial Technology May 2016The present study highlights the microbial synthesis of silver and gold nanoparticles by Sporosarcina koreensis DC4 strain, in an efficient way. The synthesized...
The present study highlights the microbial synthesis of silver and gold nanoparticles by Sporosarcina koreensis DC4 strain, in an efficient way. The synthesized nanoparticles were characterized by ultraviolet-visible spectrophotometry, which displayed maximum absorbance at 424nm and 531nm for silver and gold nanoparticles, respectively. The spherical shape of nanoparticles was characterized by field emission transmission electron microscopy. The energy dispersive X-ray spectroscopy and elemental mapping were displayed the purity and maximum elemental distribution of silver and gold elements in the respective nanoproducts. The X-ray diffraction spectroscopy results demonstrate the crystalline nature of synthesized nanoparticles. The particle size analysis demonstrate the nanoparticles distribution with respect to intensity, volume and number of nanoparticles. For biological applications, the silver nanoparticles have been explored in terms of MIC and MBC against pathogenic microorganisms such as Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, Bacillus anthracis, Bacillus cereus and Staphylococcus aureus. Moreover, the silver nanoparticles in combination with commercial antibiotics, such as vancomycin, rifampicin, oleandomycin, penicillin G, novobiocin, and lincomycin have been explored for the enhancement of antibacterial activity and the obtained results showed that 3μg concentration of silver nanoparticles sufficiently enhance the antimicrobial efficacy of commercial antibiotics against pathogenic microorganism. Furthermore, the silver nanoparticles potential has been reconnoitered for the biofilm inhibition by S. aureus, Pseudomonas aeruginosa and E. coli and the results revealed sufficient activity at 6μg concentration. In addition, gold nanoparticles have been applied for catalytic activity, for the reduction of 4-nitrophenol to 4-aminophenol using sodium borohydride and positive results were attained.
Topics: Anti-Infective Agents; Gold; Metal Nanoparticles; Microbial Sensitivity Tests; Nanotechnology; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Silver; Sporosarcina
PubMed: 26992796
DOI: 10.1016/j.enzmictec.2016.02.005 -
European Journal of Drug Metabolism and... Dec 2018Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible...
BACKGROUND AND OBJECTIVES
Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated.
METHODS
Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding.
RESULTS
Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred.
CONCLUSIONS
This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.
Topics: Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP2D6 Inhibitors; Cytochrome P-450 CYP3A Inhibitors; Dihydralazine; Dose-Response Relationship, Drug; Drug Interactions; Humans; Microsomes, Liver; Mifepristone; Paroxetine; Theophylline; Troleandomycin; Verapamil
PubMed: 29785610
DOI: 10.1007/s13318-018-0485-7 -
International Journal of Systematic and... Sep 2014A novel Gram-staining-positive, aerobic bacterium, designed DCY80(T), was isolated from soil of a ginseng field in the Republic of Korea. 16S rRNA gene sequence analysis...
A novel Gram-staining-positive, aerobic bacterium, designed DCY80(T), was isolated from soil of a ginseng field in the Republic of Korea. 16S rRNA gene sequence analysis revealed that strain DCY80(T) belonged to the genus Brachybacterium (95.8-98.2 % similarity) and was most closely related to Brachybacterium faecium DSM 4810(T) (98.2 %). Colonies were circular, entire, low-convex, opaque and 0.5-1.0 mm in diameter after growth for 2 days on TSA at 30 °C. Growth occurred at 4-34 °C (optimum, 25 °C), at pH 5.0-10.0 (optimum, pH 6.5-7.0) and in the presence of 0-7.0 % NaCl. Strain DCY80(T) produced siderophores and was sensitive to penicillin G, erythromycin, cefazolin, oleandomycin, ceftazidime, vancomycin, tetracycline, novobiocin, carbamicillin, rifampicin and neomycin. The DNA G+C content was 71.0 mol%. Levels of DNA-DNA relatedness between strain DCY80(T) and B. faecium DSM 4810(T), B. paraconglomeratum KCTC 9916(T), B. saurashtrense DSM 23186(T) and B. conglomeratum KCTC 9915(T) were 46.9±0.5, 28.9±0.6, 20.4±0.9 and 17.3±0.4 %, respectively. The cell-wall peptidoglycan of strain DCY80(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. The menaquinones were MK-7 (85.8 %) and MK-8 (14.2 %). The major cellular fatty acids were anteiso-C15 : 0 (69.1 %) and anteiso-C17 : 0 (12.2 %). Phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid, two unidentified phospholipids and five unidentified polar lipids were found. On the basis of our phenotypic and genotypic analyses, strain DCY80(T) represents a novel species of the genus Brachybacterium, for which the name Brachybacterium ginsengisoli sp. nov. is proposed (type strain DCY80(T) = KCTC 29226(T) = JCM 19356(T)).
Topics: Actinomycetales; Base Composition; Cell Wall; DNA, Bacterial; Diaminopimelic Acid; Fatty Acids; Molecular Sequence Data; Nucleic Acid Hybridization; Panax; Peptidoglycan; Phospholipids; Phylogeny; RNA, Ribosomal, 16S; Republic of Korea; Sequence Analysis, DNA; Soil Microbiology; Vitamin K 2
PubMed: 24944333
DOI: 10.1099/ijs.0.058388-0 -
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 -
The Journal of Antimicrobial... Oct 2016Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are...
BACKGROUND
Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are thought to be important factors in disease severity and duration. Macrolides including azithromycin are often used to treat the above diseases, but exhibit variable levels of efficacy. Inhaled corticosteroids are also readily used in treatment, but may lack specificity. Ideally, new treatment alternatives should suppress unwanted inflammation, but spare beneficial antiviral immunity.
METHODS
In the present study, we screened 225 novel macrolides and tested them for enhanced antiviral activity against rhinovirus, as well as anti-inflammatory activity and activity against Gram-positive and Gram-negative bacteria. Primary bronchial epithelial cells were grown from 10 asthmatic individuals and the effects of macrolides on rhinovirus replication were also examined. Another 30 structurally similar macrolides were also examined.
RESULTS
The oleandomycin derivative Mac5, compared with azithromycin, showed superior induction (up to 5-fold, EC50 = 5-11 μM) of rhinovirus-induced type I IFNβ, type III IFNλ1 and type III IFNλ2/3 mRNA and the IFN-stimulated genes viperin and MxA, yet had no effect on IL-6 and IL-8 mRNA. Mac5 also suppressed rhinovirus replication at 48 h, proving antiviral activity. Mac5 showed antibacterial activity against Gram-positive Streptococcus pneumoniae; however, it did not have any antibacterial properties compared with azithromycin when used against Gram-negative Escherichia coli (as a model organism) and also the respiratory pathogens Pseudomonas aeruginosa and non-typeable Haemophilus influenzae. Further non-toxic Mac5 derivatives were identified with various anti-inflammatory, antiviral and antibacterial activities.
CONCLUSIONS
The data support the idea that macrolides have antiviral properties through a mechanism that is yet to be ascertained. We also provide evidence that macrolides can be developed with anti-inflammatory, antibacterial and antiviral activity and show surprising versatility depending on the clinical need.
Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Antiviral Agents; Asthma; Bronchi; Cells, Cultured; Drug Discovery; Drug Evaluation, Preclinical; Epithelial Cells; Escherichia coli; Gram-Negative Bacteria; Gram-Positive Bacteria; Haemophilus influenzae; Humans; Interferon-beta; Interferons; Interleukin-6; Interleukin-8; Macrolides; Myxovirus Resistance Proteins; Oxidoreductases Acting on CH-CH Group Donors; Proteins; Pseudomonas aeruginosa; Rhinovirus; Virus Replication
PubMed: 27494903
DOI: 10.1093/jac/dkw222 -
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 -
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