Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.

Metabolome Profiling in the Plasma of Dogs with Idiopathic Dilated Cardiomyopathy: A Multiplatform Mass-Spectrometry-Based Approach.

Dilated cardiomyopathy is one of the important diseases in dogs and humans. The second most common cause of heart failure in dogs is idiopathic dilated cardiomyopathy (iDCM), which results in heart failure or sudden cardiac death due to arrhythmia. This study aimed to determine changes in the plasma metabolome of dogs with iDCM compared to healthy dogs. For that purpose, a multiplatform mass-spectrometry-based approach was used. In this study, we included two groups of dogs: 12 dogs with iDCM and 8 healthy dogs. A total of 272 metabolites were detected in the plasma samples of dogs by combining three approaches but four MS-based platforms (GC-MS, LC-MS (untargeted), LC-MS (targeted), and FIA-MS (targeted) methods). Our findings demonstrated changes in the canine plasma metabolome involved in the development of iDCM, including the different concentrations of amino acids, biogenic amines, acylcarnitines, triglycerides and diglycerides, sphingomyelins, and organic acids. The results of this study will enable the detection and monitoring of pathophysiological mechanisms involved in the development of iDCM in the future.

Multi Platforms Strategies and Metabolomics Approaches for the Investigation of Comprehensive Metabolite Profile in Dogs with Babesia canis Infection.

Canine babesiosis is an important tick-borne disease worldwide, caused by parasites of the Babesia genus. Although the disease process primarily affects erythrocytes, it may also have multisystemic consequences. The goal of this study was to explore and characterize the serum metabolome, by identifying potential metabolites and metabolic pathways in dogs naturally infected with Babesia canis using liquid and gas chromatography coupled to mass spectrometry. The study included 12 dogs naturally infected with B. canis and 12 healthy dogs. By combining three different analytical platforms using untargeted and targeted approaches, 295 metabolites were detected. The untargeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomics approach identified 64 metabolites, the targeted UHPLC-MS/MS metabolomics approach identified 205 metabolites, and the GC-MS metabolomics approach identified 26 metabolites. Biological functions of differentially abundant metabolites indicate the involvement of various pathways in canine babesiosis including the following: glutathione metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; cysteine and methionine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. This study confirmed that host-pathogen interactions could be studied by metabolomics to assess chemical changes in the host, such that the differences in serum metabolome between dogs with B. canis infection and healthy dogs can be detected with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Our study provides novel insight into pathophysiological mechanisms of B. canis infection.

Rapid Structure Determination of Bioactive 4″-Tetrahydrofurfuryl Macrozone Reaction Mixture Components by LC-SPE/Cryo NMR and MS.

LC-SPE/cryo NMR and MS methodologies have been developed and employed for a rapid structure determination of 4″-tetrahydrofurfuryl macrozone reaction mixture components. Macrozones, novel conjugates of azithromycin, and thiosemicarbazones have shown very good in vitro antibacterial activities against susceptible and some resistant bacterial strains and are promising agents for further development. The post-column multiple trapping of the chromatographically separated reaction mixture components on the SPE cartridges increased the sensitivity and together with cryogenically cooled NMR probe made it possible to identify and structurally characterize main 4″-tetrahydrofurfuryl macrozone reaction mixture compounds including those present at very low concentration level. This approach has several advantages over a classical off-line procedure, efficiency and low solvent consumption being the two most important ones. All identified components were process-related. It has been demonstrated that two different kinds of compounds with respect to structure were identified, i.e., macrolide-related and thiosemicarbazone-related ones. This methodology can serve as a platform for reliable and effective macrolides reaction components structure profiling, serving as both isolation and identification tools.

Advanced Methods for Studying Structure and Interactions of Macrolide Antibiotics.

Macrolide antibiotics are macrocyclic compounds that are clinically used and prescribed for the treatment of upper and lower respiratory tract infections. They inhibit the synthesis of bacterial proteins by reversible binding to the 23S rRNA at or near the peptidyl transferase center. However, their excellent antibacterial profile was largely compromised by the emergence of bacterial resistance. Today, fighting resistance to antibiotics is one of the greatest challenges in medicinal chemistry. Considering various physicochemical properties of macrolides, understanding their structure and interactions with macromolecular targets is crucial for the design of new antibiotics efficient against resistant pathogens. The solid-state structures of some macrolide-ribosome complexes have recently been solved, throwing new light on the macrolide binding mechanisms. On the other hand, a combination of NMR spectroscopy and molecular modeling calculations can be applied to study free and bound conformations in solution. In this article, a description of advanced physicochemical methods for elucidating the structure and interactions of macrolide antibiotics in solid state and solution will be provided, and their principal advantages and drawbacks will be discussed.

High Resolution for Chemical Shifts and Scalar Coupling Constants: The 2D Real-Time J-Upscaled PSYCHE-DIAG.

The facile determination of chemical shift and scalar coupling constants in NMR spectra is often prevented by spectral overlap and limited resolution. Here, we present a high-resolution NMR experiment for the simultaneous detection of both resonance frequencies and coupling patterns even with small J-values. A PSYCHE-decoupled DIAG (Pure Shift Yielded by Chirp Excitation- DIAGonal) experiment, which resolves chemical shift in the indirect dimension of a 2D experiment is combined with real-time J-upscaling in order to visualize small coupling constants that would otherwise be hidden in the linewidth of a regular proton or DIAG spectrum.

Structural and functional implications of the interaction between macrolide antibiotics and bile acids.

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC50 ) values.

Fast determination of diesel fuel oxidation stability by 1H NMR spectroscopy.

(1)H NMR spectroscopy and multiple linear regression analysis were used to determine diesel fuel oxidation stability. Oxidation stability is one of the most important properties which affect diesel fuel characteristics and quality. It has been demonstrated that integrated peak intensities of certain functional groups in (1)H NMR spectra correlated well with the oxidation stability. This approach is fast, nondestructive and requires small amounts of samples. It has a potential to be used as an alternative to existing standard methods.

Condensation reaction between carbohydrazide and salicylaldehyde: in-line vibrational spectroscopy monitoring and characterization of the reaction products in solution and solid state.

The condensation reaction between carbohydrazide and salicylaldehyde was monitored in-line by using vibrational NIR and Raman spectroscopies and statistical methods. Prior to in-line data analysis the reaction products were fully characterized in solution and solid state in order to check the potential of the in-line approach as a tool for in-process Schiff bases reaction control. It was demonstrated that a combination of vibrational spectroscopy and principal component analysis made it possible to detect and identify the reaction products, e.g. mono(salicylidene)carbohydrazide (1) and bis(salicylidene)carbohydrazide (2) in different solvents, and to determine the reaction end points in real time. Owing to complexity of the reaction mixtures and band overlapping, it was not possible to determine the relative ratio of the reaction products in-line. The off-line analysis showed that 1 was predominant in methanol while the highest portion of 2 was obtained in ethanol.

Deep reinforcement learning classification of sparkling wines based on ICP-MS and DOSY NMR spectra.

An approach that combines NMR spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) and advanced tensor decomposition algorithms with state-of-the-art deep learning procedures was applied for the classification of Croatian continental sparkling wines by their geographical origin. It has been demonstrated that complex high-dimensional NMR or ICP-MS data cannot be classified by higher-order tensor decomposition alone. Extension of the procedure by deep reinforcement learning resulted in an exquisite neural network predictive model for the classification of sparkling wines according to their geographical origin. A network trained on half of the sample set was able to classify even 94% of all samples. The model can particularly be useful in cases where the number of samples is limited and when simpler statistical methods fail to produce reliable data. The model can further be exploited for the identification and differentiation of sparkling wines including a high potential for authenticity or quality control.

Improvement of the Nutraceutical Profile of Brewer's Spent Grain after Treatment with Trametes versicolor.

Brewer's spent grain (BSG) is an important secondary raw material that provides a readily available natural source of nutraceuticals. It finds its largest application as animal feed and part of the human diet, while the future perspective predicts an application in the production of value-added products. In order to investigate a sustainable BSG treatment method, two BSG samples (BSG1 and BSG2) were evaluated as substrates for the production of hydrolytic (xylanase, ß-glucosidase and cellulase) and lignolytic enzymes (laccase, manganese peroxidase and lignin peroxidase) by solid-state fermentation (SSF) with Trametes versicolor while improving BSG nutritional value. The biological treatment was successful for the production of all hydrolytic enzymes and laccase and manganese peroxidase, while it was unsuccessful for the production of lignin peroxidase. Because the two BSGs were chemically different, the Trametes versicolor enzymes were synthesized at different fermentation times and had different activities. Consequently, the chemical composition of the two BSG samples at the end of fermentation was also different. The biological treatment had a positive effect on the increase in protein content, ash content, polyphenolic compounds, and sugars in BSG1. In BSG2, there was a decrease in the content of reducing sugars. Cellulose, hemicellulose, and lignin were degraded in BSG1, whereas only cellulose was degraded in BSG2, and the content of hemicellulose and lignin increased. The fat content decreased in both samples. The safety-related correctness analysis showed that the biologically treated sample did not contain any harmful components and was therefore safe for use in nutritionally enriched animal feed.

Screening of Novel Antimicrobial Diastereomers of Azithromycin-Thiosemicarbazone Conjugates: A Combined LC-SPE/Cryo NMR, MS/MS and Molecular Modeling Approach.

A well-known class of antibacterials, 14- and 15-membered macrolides are widely prescribed to treat upper and lower respiratory tract infections. Azithromycin is a 15-membered macrolide antibiotic possessing a broad spectrum of antibacterial potency and favorable pharmacokinetics. Bacterial resistance to marketed antibiotics is growing rapidly and represents one of the major global hazards to human health. Today, there is a high need for discovery of new anti-infective agents to combat resistance. Recently discovered conjugates of azithromycin and thiosemicarbazones, the macrozones, represent one such class that exhibits promising activities against resistant pathogens. In this paper, we employed an approach which combined LC-SPE/cryo NMR, MS/MS and molecular modeling for rapid separation, identification and characterization of bioactive macrozones and their diastereomers. Multitrapping of the chromatographic peaks on SPE cartridges enabled sufficient sample quantities for structure elucidation and biological testing. Furthermore, two-dimensional NOESY NMR data and molecular dynamics simulations revealed stereogenic centers with inversion of chirality. Differences in biological activities among diastereomers were detected. These results should be considered in the process of designing new macrolide compounds with bioactivity. We have shown that this methodology can be used for a fast screening and identification of the macrolide reaction components, including stereoisomers, which can serve as a source of new antibacterials.

Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae.

A series of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides, were discovered, structurally characterized and biologically evaluated. They have shown good antibacterial activity against selected Gram-positive and Gram-negative bacterial strains. These include N″ substituted 9a-(N'-carbamoyl-γ-aminopropyl)- (6a,c), 9a-(N'-thiocarbamoyl-γ-aminopropyl)- (7a,e), 9a-[N'-(ß-cyanoethyl)-N'-(carbamoyl-γ-aminopropyl)]- (9a-c, 9g) 9a-[N'-(ß-cyanoethyl)-N'-(thiocarbamoyl-γ-aminopropyl)]-derivatives (10d-f) of 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3). Among the synthesized compounds thiourea 7a and urea 9b have shown substantially improved activity comparable to azithromycin (1) and significantly better activity than the 3-decladinosyl-azithromycin (2) and the parent 3-cladinosyl analogues against efflux-mediated resistant S. pneumoniae.

Interactions of Aminopropyl-Azithromycin Derivatives, Precursors in the Synthesis of Bioactive Macrozones, with E. coli Ribosome: NMR and Docking Studies.

The structure and interactions of several aminopropyl-azithromycin derivatives (1a-c) have been studied by using NMR spectroscopy and docking calculations. Compounds 1a-c are precursors in the synthesis of macrozones, novel bioactive azithromycin-thiosemicarbazone conjugates active against some resistant bacterial strains. Today, bacterial resistance is considered as one of the major threats to human health. Knowledge on drug binding mode and conformations is one of the key factors in the process of designing molecules to fight resistance. In solution state, compounds 1a and 1c exist in the 3-endo-folded-out conformation, while 1b adopts a classical folded-out conformation. 13C and 15N CPMAS NMR spectra pointed towards similar structures in the solid state. The transferred NOESY NMR spectra confirmed binding to the E. coli ribosome and suggest that dominant conformations in the bound state resemble those in the free one. STD experiments identified reactive groups of 1a-c in close contact with the ribosome resembling binding epitopes observed for the related 15-membered macrolides. Docking studies revealed that the studied compounds bind to the same ribosome binding pocket similarly to erythromycin in the crystal state, and that the binding is achieved through H-bonds and van der Waals interactions. The bound conformation is the same as determined by NMR. STD enhancements observed for methylene protons in the aminopropyl side chain indicate additional interactions which contribute to the overall binding energy.

Discovery of macrozones, new antimicrobial thiosemicarbazone-based azithromycin conjugates: design, synthesis and in vitro biological evaluation.

Increasing bacterial resistance to existing antibiotics presents a serious threat to human health, and new antibacterial agents are desperately needed. Unfortunately, the number of newly marketed antibiotics has decreased dramatically in recent years. Withdrawal of the macrolide antibiotic telithromycin and the inability of solithromycin to gain marketing approval have prompted our efforts to search for new anti-infective macrolide compounds. Here we present the design, synthesis and biological evaluation of a novel hybrid class of azithromycin conjugates, the macrozones. Evaluation of prepared compounds against a panel of pathogenic bacteria revealed that these molecules showed excellent activities against susceptible Streptococcus pneumoniae, Streptococcus pyogenes and Enterococcus faecalis strains comparable with or better than azithromycin. Furthermore, prepared macrozones exhibited excellent activity against efflux resistant S. pneumoniae, which was 32 times better than that of azithromycin, and very good activity against an efflux resistant Staphylococcus aureus strain against which azithromycin is inactive. The results described here can serve as a good basis to guide further activities directed toward the discovery of more potent macrolide anti-infectives.

Free and bound state structures of 6-O-methyl homoerythromycins and epitope mapping of their interactions with ribosomes.

The solution and solid state conformations of several 6-O-methyl homoerythromycins 1-4 were studied using a combination of X-ray crystallography, NMR spectroscopy and molecular modelling calculations. In the solid state 1 was found to exist as the two independent molecules with similar structures termed 3-endo-folded-out. In solution a significant conformational flexibility was noticed especially in the C2 to C5 region. The compounds 1 and 2 unlike 14-membered macrolides adopted the 3-endo-folded-out conformation while 3 and 4 existed in the classical folded-out conformation. TrNOESY and STD experiments showed that 1 and 2 bound to the Escherichia coli ribosome while 3 and 4, lacking the cladinose sugar, did not exhibit binding activities, this being in accordance with biochemical data. The bound conformations were found to be very similar to the free ones, some small differences were observed and discussed. The STD experiments provided evidence on binding epitopes. The structural parts of 1 and 2 in close contact with ribosome were similar, however the degree of saturation transfer was higher for 2. The differences between tr-NOE data and STD enhancements in 1 and 2 arouse as a consequence of structural changes upon binding and a closer proximity of 2 to the ribosome surface. An understanding of the molecular mechanisms involved in the interaction of macrolides with ribosomes can help in developing strategies aiming at design of potential inhibitors.

16-membered macrolide antibiotics: a review.

The 16-membered macrolide antibiotics (e.g. tylosin A and josamycin) are mainly used in veterinary medicine, and are much less studied than their 14- and 15-membered erythromycin-based cousins. Although these antibiotics have similar antibacterial profiles, with activity primarily against Gram-positive and a limited range of Gram-negative organisms, the 16-membered macrolides show some advantages. These include better gastrointestinal tolerance, lack of drug-drug interactions, and activity against certain resistant bacterial strains by extension of the peptide tunnel reach allowing additional interactions. In addition to antibacterial activity, the most famous representative of the class, tylosin A, as well as some derivatives of desmycosin (tylosin B), have shown antimalarial activity. Such activity has also been observed in the 14-membered macrolide antibiotics, azithromycin, solithromycin and clindamycin. This antimalarial activity provides the opportunity to investigate these drugs as cheap and effective antimalarials. This is an overview of the latest research on biosynthesis, structure, chemical properties and mode of action of 16-membered macrolides, with special emphasis on their most explored members: tylosin A and josamycin.

Azithromycin-sulfonamide conjugates as inhibitors of resistant Streptococcus pyogenes strains.

Novel hybrid compounds 6a-6d, conjugates of 15-membered azalides and sulfonamides, i.e. unsubstituted, 4-aryl- and 4-heteroaryl-aminosulfonyl derivatives of 9a-[N'-(phenylcarbamoyl)]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A were synthesized and characterized by IR, one- and two-dimensional NMR spectroscopies and MALDI-TOF and MS/MS mass spectrometry. The new compounds were evaluated in vitro against a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. 9a-{N'-[4-(Aminosulfonyl)phenyl]carbamoyl}--(6a) and 9a-{N'-[4-(phenylaminosulfonyl)phenyl]carbamoyl}--(6b) derivatives showed improvements in activity against inducible resistant Streptococcus pyogenes in comparison with macrolide antibiotic azithromycin and starting material 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2). In addition, the synthesized azithromycin-sulfonamide conjugates 6a-6d showed good antibacterial activity against sensitive S. pyogenes and Streptococcus pneumoniae strains. The kinetics of degradation in the artificial gastric juice showed that the most active compounds, 6a and 6b, exhibited azithromycin like stability. The cleavage of the cladinose sugar was found to be the main decomposition pathway leading to inactive 7a and 7b, prepared also as analytical standards by the alternative synthetic route together with 7c and 7d.

The application of LC-NMR and LC-MS for the separation and rapid structure elucidation of an unknown impurity in 5-aminosalicylic acid.

This work has demonstrated the usefulness of combining liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR) and liquid chromatography-mass spectrometry (LC-MS) methodologies for a rapid identification of an unknown impurity (N1) in the drug 5-aminosalycilic acid. Complementary information obtained from the two methods has revealed plenty of structural information and led to the fast on-line structure determination of N1 prior to its isolation and purification. The analysis of LC-NMR and LC-MS spectra revealed that N1 and 5-aminosalycilic acid are structurally closely related compounds. The structure of N1 was later confirmed by high-resolution NMR spectroscopy of the isolated compound and the atom assignment was made. The approach described here has potential for 5-aminosalycilic acid impurity profiling and monitoring the production process.

Synthesis, characterization and in vitro antimicrobial activity of novel sulfonylureas of 15-membered azalides.

Three series of the novel sulfonylurea derivatives of 15-membered azalides, i.e. 9a-N-[N'-(aryl)sulfonylcarbamoyl] (4a-4f, 5a-5f), 9a-N-{N'-[(aryl)sulfonylcarbamoyl-gamma-aminopropyl]} (10a-10f, 11a, 11c) and 9a-N-{N'-(beta-cyanoethyl)-N'-[(aryl)sulfonylcarabamoyl-gamma-aminopropyl]} (14a-14f, 15a, 15b, 15f) derivatives of 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2) and 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3) were prepared and their structures elucidated by NMR and IR spectroscopic methods and mass spectrometry. Minimal inhibitory concentration (MIC) of these compounds was determined on a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. Several compounds of the series of 9a-N-[N'-(aryl)sulfonylcarbamoyl] derivatives that showed significant improvements in activity against inducible resistant Streptococcus pyogenes strain were suggested for further optimization.