Binding Of Tetracyclines Research Articles

Two-Photon Excited Fluorescence Lifetime Imaging of Tetracycline-Labeled Retinal Calcification.

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79-97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board.

Broad-Spectrum Detection of Tetracyclines by Riboswitch-Based Cell-Free Expression Biosensing.

We describe a sensitive and selective method for the determination of tetracycline content in foods using a riboswitch sensor. The sensor is based on a cell-free expression system that can be lyophilized to produce paper-based sensors or tube-based sensors for long-term storage. The riboswitch constructed using artificially screened tetracycline RNA aptamers was cloned into the pET-28a(+) vector of Escherichia coli TOP 10. The expression of the green fluorescent protein was positively correlated with the concentration of tetracyclines. The binding of tetracyclines to the aptamer domain results in a conformational change in the riboswitch secondary structure, resulting in the exposure of the ribosome binding site thereby promoting expression. The detection limits of the prepared sensor for the detection of tetracycline, oxytetracycline, chlortetracycline, and doxycycline were 0.47, 0.079, 0.084, and 0.43 μM, respectively. Moreover, the 1 μM tetracyclines allow for qualitative detection in milk samples by the naked eye. The work provides a proof-of-principle for riboswitch design to address global health and food safety.

Selective recognition of tetracycline residues in animal derived samples based on molecularly imprinted microspheres from silica-stabilised Pickering emulsion polymerisation

ABSTRACT Novel molecularly imprinted polymers for selective binding of tetracyclines were synthesised through oil-in-water Pickering emulsion polymerisation with SiO2 particles as stabilisers. The products were used for solid phase extraction coupled with high-performance liquid chromatography to detect trace tetracycline and oxytetracycline in animal-derived samples. The SPE materials were characterised in detail by Fourier transform infrared spectrometry, scanning electron microscopy and thermogravimetry analysis. The imprinted polymers displayed high adsorption capacity, fast binding process and high selectivity through evaluation of adsorption performance employing kinetic, static adsorption and selectivity experiments. Under optimum detection conditions, good linearity (12 to 121 µg kg−1) and limits of detection (1.8 to 1.9 μg kg−1) for tetracyclines were obtained. In addition, satisfactory recoveries of 75% to 115 % were also achieved by spiking tetracyclines into milk, chicken, fish and pork samples, giving direct evidence of real practicality of our proposed separation method.

Effect of iron chelation on anti-pseudomonal activity of doxycycline

BackgroundIncreasing resistance of microorganisms to antimicrobial agents is a growing concern and there is a lack of novel agents. This has stimulated the exploration of novel strategies for treatment of infection. ObjectiveTo investigate synergistic interactions between five tetracyclines and tobramycin with an iron chelator (CP762) against two reference strains and nine clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients. MethodMicrodilution assays for minimal inhibitory concentration determination and checkerboard assays were used to assess synergy between antibiotics and CP762. Given the iron-binding capacity of tetracyclines, the binding of iron with doxycycline was investigated using Job's plot methodology. Synergy between the iron-bound form of doxycycline and CP762 was compared with that of unbound doxycycline and CP762. Enhancement of doxycycline anti-biofilm activity was also assessed. ResultsThere was synergy between CP762 and all tetracyclines, except minocycline, against the reference strains but that against clinical isolates was variable. Synergy was not demonstrated for tobramycin against any of the strains tested. This led to the hypothesis that iron chelation preserves the binding of tetracyclines to the bacterial ribosome. Susceptibility to iron-bound doxycycline was decreased by two- to four-fold and synergistic interactions with the iron chelator were consistently more intense with iron-bound doxycycline than with doxycycline alone. The doxycycline–iron chelator combination also significantly reduced cell viability in established biofilms. ConclusionThe data in this study provide evidence that iron chelation enhances the anti-pseudomonal activity of tetracyclines, specifically doxycycline.

Binding of Tetracyclines to Acinetobacter baumannii TetR Involves Two Arginines as Specificity Determinants.

Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.

The complexity of minocycline serum protein binding.

Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines.

Determination of tetracyclines in meat using two phases freezing extraction method and HPLC-DAD

The protein binding of tetracyclines may cause the common extraction methods are not able to completely remove them from the proteins. To overcome this problem, we introduced a simple, reliable, sensitive, and reproducible method for simultaneous clean-up and preconcentration of 4 tetracyclines in chicken meats based on two phase freezing method.

Estimation of the degree of binding of tetracyclines in human plasma.

Estimation of the degree of binding of tetracyclines in human plasma.

Binding of Tetracyclines to Elongation Factor Tu, the Tet Repressor, and the Ribosome: A Molecular Dynamics Simulation Study

Tetracycline (Tc) is a broad-spectrum antibiotic that kills bacteria by interrupting protein biosynthesis. It is thought that the bacteriostatic action of Tc is associated with its binding to the acceptor site (or A site) in the bacterial ribosome, interfering with the attachment of aminoacyl-tRNA. Recently, however, the crystal structure of a complex between Tc and trypsin-modified elongation factor Tu (tm-EF-Tu) was determined, raising the question of whether Tc binding to EF-Tu has a role in its inhibition of protein synthesis. We address this question using computer simulations. As controls, we first compute relative ribosome binding free energies for seven Tc variants for which experimental data are available, obtaining good agreement. We then consider the binding of Tc to both the trypsin-modified and unmodified EF-Tu-GDP complexes. We show that the direct contribution of EF-Tu to the binding free energy is negligible; rather, the binding can be solely attributed to interactions of Tc with a bridging Mg(2+) ion and the GDP phosphate groups. The effects of trypsin modification are modest. Further, our calculations show that EF-Tu does not exhibit any binding preference for Tc over the nonantibiotic, 4-dedimethyl-Tc, and EF-Tu does not bind the Tc analogue tigecycline, which is a potent antibiotic. In contrast, both the ribosome and the Tet Repressor protein (involved in Tc resistance) do show a binding preference for Tc over 4-dedimethyl-Tc, and the ribosome prefers to bind tigecycline over Tc. Overall, our results provide insights into the binding properties of tetracyclines and support the idea that EF-Tu is not their primary target.

Interactions of tetracycline and its derivatives with DNA in vitro in presence of metal ions

The interactions of calf thymus DNA with tetracycline (TC), 7-chlorotetracycline (CTC) and 6-dimethyl-7-chlorotetracycline (DMTC) were assessed employing spectrofluorometric and circular dichroism (CD) techniques. The Scatchard analysis revealed relatively lesser binding affinity of TC ( K a=1.2×10 7 l mol −1) vis-a-vis CTC ( K a=3.4×10 7 l mol −1) and DMTC ( K a=3.0×10 7 l mol −1) with DNA. The data suggested both the intercalative and electrostatic nature of binding between the tetracyclines and DNA. The presence of Cu(II) augmented the interaction of tetracyclines with DNA, and resulted in red shift by 12 nm in CD spectra of tetracycline. The molar ellipticity ( θ) also changed significantly for CTC and DMTC. The data unequivocally demonstrated the DNA binding potential of tetracyclines both in the presence and absence of Cu(II) ions in dark. The enhanced binding of tetracyclines in presence of Cu(II), ensuing conformational changes in DNA secondary structure to a varying extent, reflects differential reactivity of ligand chromophores.

The bacterial ribosome, a promising focus for structure-based drug design.

Recent crystal structures of the bacterial ribosome have identified the complex molecular interactions involved in antibiotic-ribosome recognition. Insights into the binding of aminoglycosides, macrolides, tetracyclines and other antibiotics provide opportunities for computational, structure-based approaches to be used in the design of appropriate modifications to existing antibiotics as well as in the discovery of completely new drug classes.

Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein

Interaction of tetracycline (TC) derivatives viz. oxytetracycline, doxycycline, demeclocycline and chlorotetracycline with bovine serum albumin (BSA) and concomitant changes in protein conformation were studied using fluorescence quenching and circular dichroism measurements. Fluorescence data revealed the presence of one to three binding sites on BSA for different TC derivatives. Binding studies with the marker ligands, warfarin and bilirubin, elucidated site-I as a primary binding site for TCs on albumin. Scatchard analysis revealed the binding affinity ( K a) and capacity ( n) for these derivatives vary in the range from 0.8 to 3.2×10 6 l/mole and 1.3–3.4, respectively. Significant reduction (60–45%) in secondary structure (α-helical content) of BSA was noticed upon interaction with different TC derivatives in presence of Cu (II) ions. High affinity binding of TCs with BSA signifies drug stability. However, excessive binding at higher TC concentrations in combination with Cu (II) induces conformational change in protein structure, which may exert detrimental effect on cellular protein.

Tet repressor-tetracycline interaction.

Previous studies [Wasylewski et al. (1996), J. Protein Chem. 15, 45-58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7 x 10(9) M-1 sec-1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%). Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg.tc]+ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.

Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis.

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

Some observations on the adsorption of tetracyclines to glass and plastic labware

The binding of six tetracyclines to borosilicate glass or polypropylene test tubes was studied in vitro, and included chlorotetracycline, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline. All tetracyclines bound to borosilicate glass test tubes, and minocycline bound the most. Both time for binding to occur, and exposure to increasing amounts of surface area were involved in the production of increased binding, but the binding was mostly due to surface area. Doxycycline and minocycline significantly bound to polypropylene test tubes, whereas all of the other tetracyclines did not. This study suggests that all containers should be tested for possible binding of tetracyclines before use, and polypropylene labware is less likely to bind tetracyclines, compared to borosilicate glass.

Binding study of tetracyclines to human serum albumin using difference spectrophotometry

The binding of several tetracyclines to human serum albumin was studied using difference spectrophotometry and a spectrophotometric probe, 2‐(4′‐hydroxybenzeneazo)benzoic acid. Difference spectra observed for the interaction between the probe and human serum albumin were similar to probe—bovine serum albumin spectra but were less intense for a given concentration of probe and did not reach saturation as quickly. Difference spectra for the tetracyclines were dependent on the characteristics of the ring substituents. More hydrophobic substituents on the D and C rings tended to give more intense difference spectra, but charge‐transfer complexing may also have been involved since methacycline with a methylene group in the 6‐position showed the most intense spectra of the compounds studied. Solvent perturbation, pH, and urea studies tended to confirm that something other than hydrophobic binding of the tetracyclines was involved. Drug‐probe displacement studies showed that methacycline gave the greatest probe displacement followed by doxycycline, chlortetracycline, oxytetracycline, and tetracycline. This order of displacement of the anionic probe indicates that both hydrophobic and charge‐transfer binding are involved. Experiments with calcium ion and ethylenediaminetetraacetic acid showed that the difference spectra obtained with the tetracyclines and human serum albumin were not the result of metallic bridge—chelate formation.

Fluorometric study of tetracycline-bovine serum albumin interaction: The tetracyclines—A new class of fluorescent probes

The interaction of four tetracyclines (tetracycline, oxytetracycline, chlortetracycline and methacycline) with bovine serum albumin is studied fluorometrically. It is demonstrated that these tetracyclines exhibit the main features of fluorescent probes; increase of their fluorescence and quantum yield both in mixtures with bovine serum albumin and in non-polar, organic solvents, accompanied by blue shift of fluorescence emission maximum. The thermodynamic values ΔH and ΔS of the reaction of binding of tetracyclines to bovine serum albumin have been determined fluorometrically. The results reveal that the binding of tetracyclines to bovine serum albumin is due mainly to non-polar (hydrophobic) interactions. It has been found that the divalent metal ions are not involved in the binding of tetracyclines to bovine serum albumin at least at pH 10·5.

Binding of tetracyclines to bone.

TETRACYCLINE (I) (ref. 1) and its derivatives oxytetracycline, chlortetracycline and (1-pyrrolidinylmethyl)-tetracycline2 are taken up in newly-formed bone after injection into the living organism, to form a zone that is intensely fluorescent under ultra-violet light. This reaction, which occurs wherever there is active deposition of new bone3, can be used for the detection of calcification. Because mineralized dead bone also takes up tetracycline on every surface (including, although to a lesser extent, the non-growing surfaces)4, the reaction does not depend on the activity of living bone cells or on the integrity of the physical properties of the intercellular components. Instead, there appears to be a direct reaction between tetracycline and one or more inorganic components of the mineralized tissue, the most likely species being hydroxyapatite, Ca10(PO4)6(OH)2, which is the major constituent of all such tissue5. A plausible suggestion as to the nature of the bonding involved is now offered.

ENHANCEMENT OF ANTIMICROBIAL ACTIVITY OF PENICILLINS AND OTHER ANTIBIOTICS IN HUMAN SERUM BY COMPETITIVE SERUM BINDING INHIBITORS.

SummaryA series of compounds, many of which had been shown in dialysis studies to be effective in displacing penicillins from binding to human serum, have been tested for ability to enhance the antimicrobial activity of various penicillins and other antibiotics in the presence of 50% human serum. Enhanced antimicrobial activity in serum was shown in the case of sulfonamides to be only partially reversible by para-amino ben-zoic acid, suggesting that augmentation of penicillin activity was due to serum displacing effects as well as to the intrinsic antibacterial activity of sulfonamides. Evidence is presented that the “R” groups of penicillins are the most important determinants of binding to serum. The specificity of the penicillin binding sites was shown by the failure of displacing agents shown to be effective against the penicillin site(s), to effect binding of novobiocin and tetracyclines, and by the lack of effect of probenecid, known to be highly bound, to displace penicillins from serum.The author ...

In vitro Binding of Tetracyclines to Calcium

SINCE antibiotics of the tetracycline series were first demonstrated to localize and persist in vivo specifically in newly proliferated bone and in certain areas of calcified cartilage1, it appears to have been generally accepted that these antibiotics do in fact bind to calcium ions in at least crystal-seeded nucleation sites on collagen fibrils and in other physiologically ‘available’ calcium-impregnated protein matrices. Nevertheless, some little controversy seems to have arisen recently owing both to the presence of diffuse though minimal localization of tetracycline fluorescence in non-growing skeletal areas2 and, as cited in the original communications, the apparent, often transient, localization of these drugs in certain grossly non-calcified soft tissues.