Micellar liquid chromatography with dodecyl dimethyl betaine as an in vitro method for prediction of protein-drug binding

With the accelerating development of new drugs, there is a high need for rapid and simple screening technologies. In this paper, a new in vitro method, dodecyl dimethyl betaine (BS-12) micellar liquid chromatography (MLC) was presented for prediction of protein-drug binding based on the similar property of BS-12 micelles to protein. The predictive possibility of this method was validated by comparing the retention factors of drugs (antidiabetic and antibacterial drugs) on C18 modified by different surfactants with those on the protein column. Through the investigation of the concentration and pH effect on the retention of the drugs in BS-12 MLC, quantitative retention-protein binding relationships were established according to the retention factors in 0.2 M BS-12 (pH 7.4) MLC and those on the protein column. According to the relationships established, the protein binding of seven drugs for psychiatric disorders, six potential drugs for antibiotics and four commercial antibiotics were predicted. The results were consistent with those on the BSA column very well. This indicated, BS-12 MLC was a simple, fast and reproducible method to predict protein-drug binding.     

Binding of aminoglycoside antibiotics to the small ribosomal subunit: a continuum electrostatics investigation

The binding of paromomycin and similar antibiotics to the small (30S) ribosomal subunit has been studied using continuum electrostatics methods. Crystallographic information from a complex of paromomycin with the 30S subunit was used as a framework to develop structures of similar antibiotics in the same ribosomal binding site. Total binding energies were calculated from electrostatic properties obtained by solution of the Poisson-Boltzmann equation combined with a surface area-dependent apolar term. These computed results showed good correlation with experimental data. Additionally, calculation of the ribosomal electrostatic potential in the paromomycin binding site provided insight into the electrostatic mechanisms for aminoglycoside binding and clues for the rational design of more effective antibiotics.     

Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

A membrane protein of relative molecular mass (Mr) 127,000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and beta-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of Mr 127,000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127,000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127,000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5/5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for beta-lactam antibiotics and small peptides of Mr 127,000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.     

Studies on Interactions of Antibiotics with Serum Albumin by Surface Plasmon Resonance Biosensor

Introduction Humanserumalbumin(HSA)isawell known transportproteinforavarietyofmoleculesandions[1].Thebindingofadrugtoserumalbuminhasimportant pharmacokineticconsequencesbecauseitinfluences distribution,excretionandpharmacologicaleffectsof thedruginthebody…     

Synthesis and characterization of D-Alanyl-D-Alanine-Agarose

A new affinity adsorbent, using D-alanyl-D-alanine as ligand, has been prepared. The dipeptide immobilized on Activated CH-Sepharose 4B (D-Ala-D-Ala-AGA) bioselectively binds the glycopeptide antibiotics teicoplanin, vancomycin, ristocetin A (vancomycinlike group of antibiotics) while it does not bind other antibiotics equally active on cell wall biosynthesis but with different target sites, such as penicillin G, cephalosporin C, gardimycin, and bacitracin. Teicoplanin, vancomycin, and ristocetin A have similar binding characteristics for the immobilized dipeptide, as indicated by equilibrium binding experiments. The affinity constants of the three antibiotics for D-Ala-D-Ala-AGA is of the same order of magnitude (10⁵ L mol^-1) and the number of effective binding sites is similar for each antibiotic (6–7 μEq/mL of gel). The adsorption is biospecific as no binding has been observed to immobilized L-alanyl-L -alanine. D-Ala-D-Ala-AGA has been successfully used to purify teicoplanin from mixtures of different complexity and for concomitant extraction and purification from fermentation liquors by both batch adsorption and column chromatography. The antibiotic can be recovered from the resin in high yields by elution at pH 11.     

A carrier protein strategy yields the structure of dalbavancin

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic-target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier-target-antibiotic complex. Using native chemical ligation, we have linked the Lys-D-Ala-D-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier-target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-D-Ala-D-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development.     

Preparation and performance analysis of monodisperse glycidyl methacrylate modified restricted access media-imprinted materials

Using monodisperse poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) as the matrix, and pefloxacin template molecules, a novel restricted-access medium molecularly imprinted polymers with Bovine serum albumin crosslinked on its surface was prepared through reversible addition–fragmentation chain-transfer polymerization. Then, the obtained material was employed in dispersive solid-phase extraction to analyze the fluoroquinolones in untreated egg samples by high-performance liquid chromatography–ultraviolet detection. Adsorption performance revealed a good binding amount (40.72 mg/g), fast binding kinetics (25 min), satisfactory selectivity, and good ability to eliminate matrix interference. The reusability experiments indicated the materials have good reusable performance after repetition. Under the optimized conditions, restricted access media-molecularly imprinted polymers-dispersive solid-phase extraction was combined with high-performance liquid chromatography–ultraviolet to enrich fluoroquinolones in untreated eggs, good limit of detection (1.31–3.15 μg/L) and high recovery (89.5–96.8%) were obtained. The results showed that the prepared restricted-access material is promising for the direct detection of antibiotics in complex samples.     

Spectrophotometric determination of amikacin,kanamycin, neomycin and tobramycin

A spectrophotometric method for determination of neomycin, kanamycin, tobramycin and amikacin is proposed, based on the Rimini test with disodium pentacyanonitrosylferrate(II) for primary and secondary aliphatic amines. The absorbance of the coloured addition product is measured. Beer's law is valid over a wide concentration range. The method is relatively fast and can be used in control of the manufacture of the antibiotics and their purity, instead of the much slower microbiological method. It is also applicable for determination of these antibiotics in formulations.     

Cooperativity and anti-cooperativity between ligand binding and the dimerization of ristocetin A: asymmetry of a homodimer complex and implications for signal transduction

Background: Recent work has indicated that dimerization is important in the mode of action of the vancomycin group of glycopeptide antibiotics. NMR studies have shown that one member of this group, ristocetin A₁ forms an asymmetric dimer with two physically different binding sites for cell wall peptides. Ligand binding by ristocetin A and dimerization are slightly anti-cooperative. In contrast, for the other glycopeptide antibiotics of the vancomycin group that have been examined so far, binding of cell wall peptides and dimerization are cooperative.Results: Here we show that the two halves of the asymmetric homodimer formed by ristocetin A have different affinities for ligand binding. One of these sites is preferentially filled before the other, and binding to this site is cooperative with dimerization. Ligand binding to the other, less favored half of the dimer, is anti-cooperative with dimerization.Conclusions: In dinner complexes, anti-cooperativity of dimerization upon ligand binding can be a result of asymmetry, in which two binding sites have different affinities for ligands. Such a system, in which one binding site is filled preferentially, may be a mechanism by which the cooperativity between ligand binding and dimerization is fine tuned and may thus have relevance to the control of signal transduction in biological systems.     

Origins of cell selectivity of cationic steroid antibiotics

A key factor in the potential clinical utility of membrane-active antibiotics is their cell selectivity (i.e., prokaryote over eukaryote). Cationic steroid antibiotics were developed to mimic the lipid A binding character of polymyxin B and are shown to bind lipid A derivatives with affinity greater than that of polymyxin B. The outer membranes of Gram-negative bacteria are comprised primarily of lipid A, and a fluorophore-appended cationic steroid antibiotic displays very high selectivity for Gram-negative bacterial membranes over Gram-positive bacteria and eukaryotic cell membranes. This cell selectivity of cationic steroid antibiotics may be due, in part, to the affinity of these compounds for lipid A.     

Modification and inhibition of vancomycin group antibiotics by formaldehyde and acetaldehyde

It is shown that several vancomycin group antibiotics (vancomycin, eremomycin, and avoparcin) undergo spontaneous chemical modifications when kept at room temperature at neutral pH in aqueous solutions containing traces of formaldehyde or acetaldehyde. This chemical modification predominantly results in a mass increase of 12 Da in the reaction with formaldehyde and 26 Da in the case of acetaldehyde. By using tandem mass spectrometry the modification can unambiguously be identified as originating from the formation of a ring-closed 4-imidazolidinone moiety at the N-terminus of the glycopeptide antibiotics, that is, near the receptor binding pocket of the glycopeptide antibiotics. Bioaffinity mass spectrometry shows that this ring-closure results in a dramatically decreased affinity for the peptidoglycan-mimicking D-alanyl-D-alanine receptor. Additionally, in vitro inhibition measurements on two different strains of bacteria have revealed that the modified antibiotics display reduced antibacterial activity. The ring-closure is also shown to have a dissociative effect on the dimerization of the vancomycin-analogue eremomycin. The spontaneous reaction of vancomycin with formaldehyde or acetaldehyde may have implications not only for the clinical use of this class of antibiotics, but also for the effectiveness of these antibiotics when they are used in chiral separation chromatography or capillary electrophoresis.     

β-Lactam ring stability and antibacterial potency: A novel eigenvalue problem

Low inoculum potency data in vitro for 16 clinical β-lactam antibiotics have been analyzed, and a physical model for interpreting the results of a number of bacterial strains has been derived. An analytic criterion for performing a unitary transformation on the potency data is developed following the identification of a physical vector present within the data which is attributable to an activation energy required for the transport of the β-lactam into a biological membrane. This vector has inverse slope relations in Gram positive and Gram negative bacteria and provides the basis for the analytic criterion for the unitary transformation. Compounds with similar potency spectra which differ only in the absolute magnitude of their effect will possess similar transport properties. It is shown that a slow rate of membrane entry for the β-lactam has overriding consequences on differences in fast rates of binding to the target enzymes and to β-lactamases, and a second primary vector is established directly from the biological data related to the ease of β-lactam ring opening. This vector offers precise evidence for testing the solvational and theoretical requirements for predicting the biological stability of novel β-lactam ring compounds.     

19F NMR in the measurement of binding affinities of chloroeremomycin to model bacterial cell-wall surfaces that mimic VanA and VanB resistance

Background: The emergence of bacteria that are resistant to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus, has made the study of the binding characteristics of glycopeptides to biologically relevant depsipeptides important. These depsipeptides, terminating in -d-alanyl-d-lactate, mimic the cell-wall precursors of resistant bacteria.Results: The use of ¹⁹F-labelled ligands in the study of the therapeutically important vancomycin series of antibiotics is demonstrated. The substantial simplification of spectra that occurs when such labelled ligands are employed is used in the measurement of binding affinities of depsipeptides to chloroeremomycin (CE). Large enhancements of binding affinities are found at a model bacterial cell-wall surface (constituted from depsipetides that are anchored into vesicles) relative to those measured in free solution.Conclusions: Surface-enhanced binding, previously shown for strongly dimerising glycopeptide antibiotics to normal -d-alanyl-d-alanine-terminating cell-wall precursors, is now demonstrated for CE to the surface of models of VanA- and VanB-resistant bacteria. The effect of depsipeptide chain length is shown to be critically important in producing and maximising this enhancement.     

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.     

The Long-term Release of Antibiotics From Monolithic Nonporous Polymer Implants for Use as Tympanostomy Tubes

A technology is elaborated for the fabrication of a novel tympanostomy tube (TT) from solidified polymer melts (Elvax and Polyurethane) and antibiotics (Ciprofloxacin and Usnic acid) for insertion into tympanic membrane (ear drum) according to the established surgical procedure. The long-term in vitro release kinetics of the antibiotics into liquid water has been assessed using standard methods. The measured kinetic curves revealed two stages of antibiotic release into the finite space. During the first stage (fast), the fast release rate is almost invariant and is determined by the diffusion through the steady diffusion layer formed due to solution agitation. In this first stage, the influence of the initial internal transport is weak because it takes place at negligibly small distance from interface and accordingly, at negligibly concentration drop. After the antibiotic concentration decreases within the much broader layer of matrix near interface, the internal transport becomes important. This manifests itself as the second stage in measured kinetics of release curves which is characterized by a gradual decrease in rate. The minimum inhibition concentrations of three antibiotics/antimicrobial compounds for four bacterial species were measured. The first stage of fast release from the polymer implant lasts 6 days at a polymer loading by Ciprofloxacin (0.03 g/cm(3)) and this was sufficient for preventing biofilm formation on the surface of the implant material. The measured kinetic curves of drug release showed more rapid decrease in the release rate compared to the Higuchi approximation. Comparison with existing theories, which account for the finite rate of drug dissolution, showed that this may explain the observed deviation from the diffusion-controlled Higuchi model. Large dimensions of drug particles and their aggregation retard the dissolution stage and consequently the release rate. Melt blending was found to cause the drug particle aggregation within polymer matrixes which was confirmed by microscopic reexamination of the polymer implant materials.     

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl‐Acyl Carrier Protein Binding Site of FabI

The enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus. By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development.     

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.     

Highly Emissive Metal-Organic Frameworks for Sensitive and Selective Detection of Nitrofuran and Quinolone Antibiotics

The overuse of antibiotics makes its detection very significant for human health. New facile methods and high-performance sensory materials will be urgently needed for detection of antibiotics. Unfortunately, there are few reports on fluorescence enhancement of antibiotics detection. Herein, based on the modulability of the coordination mode, we proposed two MOFs with different coordination modes based on different metal ions: Zn-MOF ( 1 ) and Cd-MOF ( 2 ). The fluorescence of 1 and 2 can be efficiently and selectively quenched by nitrofuran antibiotics (nitrofurazone, NFZ and furazolidone, FZD) and chloramphenicol (CAP), respectively. Particularly, the matched energy levels between 2 and enrofloxacin (ENR) enables 2 with turn-on sensing for ENR. Moreover, apart from the sensitivity and selectivity, 1 and 2 also have strong recyclable ability, fast response time and anti-interference ability, which make them great potential sensory materials to detect antibiotics.     

Fast screening procedure for antibiotics in wastewaters by direct HPLC-DAD analysis

Growing concern about the contamination of wastewaters by antibiotics demands fast but sensitive analytical methodologies, for the screening of a large number of samples. The purpose of this work was to develop a simple methodology, using direct injection of the samples, by HPLC with diode array detection (DAD), for a multiresidue analysis of five antibiotics of different classes. Wastewater from an urban water treatment plant was selected as a model to study possible coelution of interfering compounds. The linearity interval ranged from 40 to 400 microg/L for amoxicillin (Amox), metronidazole (Metro), cefazolin (Cefa), and chloramphenicol (Chloram) and from 20 to 200 microg/L for sulfamethoxazole (Sulfa), with LODs lower than 14 microg/L. Repeatability, expressed by the CV of six repeated injections, ranged from 1 to 8%, while the intermediate precision varied between 2 and 11%. The recovery ranged from 90 to 109%. This method enables the fast screening of a large number of samples, with an expanded uncertainty in the 1-22% range. The advantage of the proposed method is to significantly reduce the number of samples to be analyzed by more complex methods.