Heterodimeric Rifampicin-Tobramycin conjugates break intrinsic resistance of Pseudomonas aeruginosa to doxycycline and chloramphenicol in vitro and in a Galleria mellonella in vivo model.

Intrinsic resistance in Pseudomonas aeruginosa, defined by chromosomally encoded low outer membrane permeability and constitutively over-expressed efflux pumps, is a major reason why the pathogen is refractory to many antibiotics. Herein, we report that heterodimeric rifampicin-tobramycin conjugates break this intrinsic resistance and sensitize multidrug and extensively drug-resistant P. aeruginosa to doxycycline and chloramphenicol in vitro and in vivo. Tetracyclines and chloramphenicol are model compounds for bacteriostatic effects, but when combined with rifampicin-tobramycin adjuvants, their effects became bactericidal at sub MIC levels. Potentiation of tetracyclines correlates with the SAR of this class of drugs and is consistent with outer membrane permeabilization and efflux pump inhibition. Overall, this strategy finds new uses for old drugs and presents an avenue to expand the therapeutic utility of legacy antibiotics to recalcitrant pathogens such as P. aeruginosa.

Hybrid Antibiotic Overcomes Resistance in P. aeruginosa by Enhancing Outer Membrane Penetration and Reducing Efflux.

Therapeutic interventions to treat multidrug-resistant (MDR) Pseudomonas aeruginosa infections are severely limited and often require the use of colistin as drug of last resort. The major challenges impeding the development of novel antipseudomonal agents are the lack of cell penetration and extensive efflux. We have discovered a tobramycin-moxifloxacin hybrid core structure which enhances outer membrane permeability and reduces efflux by dissipating the proton motive force of the cytoplasmic membrane in P. aeruginosa. The optimized hybrid protects Galleria mellonella larvae from the lethal effects of MDR P. aeruginosa. Attempts to select for resistance over a period of 25 days resulted in a 2-fold increase in the minimal inhibitory concentration (MIC) for the hybrid, while moxifloxacin or tobramycin resulted in a 16- and 512-fold increase in MIC. Although the hybrid possesses potent activity against MDR, P. aeruginosa isolates the activity that can be synergized when used in combination with other classes of antibiotics.

Singly modified amikacin and tobramycin derivatives show increased rRNA A-site binding and higher potency against resistant bacteria.

Semisynthetic derivatives of the clinically useful aminoglycosides tobramycin and amikacin were prepared by selectively modifying their 6'' positions with a variety of hydrogen bond donors and acceptors. Their binding to the rRNA A-site was probed using an in vitro FRET-based assay, and their antibacterial activities against several resistant strains (e.g., Pseudomonas aeruginosa, Klebsiella pneumonia, MRSA) were quantified by determining minimum inhibitory concentrations (MICs). The most potent derivatives were evaluated for their eukaryotic cytotoxicity. Most analogues displayed higher affinity for the bacterial A-site than the parent compounds. Although most tobramycin analogues exhibited no improvement in antibacterial activity, several amikacin analogues showed potent and broad-spectrum antibacterial activity against resistant bacteria. Derivatives tested for eukaryotic cytotoxicity exhibited minimal toxicity, similar to the parent compounds.

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate.

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.

Electrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity.

Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.

Collisionally activated dissociations of aminocyclitol-aminoglycoside antibiotics and their application in the identification of a new compound in tobramycin samples.

Several aminocyclitol-aminoglycoside antibiotics have been studied by tandem mass spectrometry. Glycosidic bond cleavages were the major reactions in the low energy collisionally activated decomposition (CAD) of the protonated antibiotics. Only the glycoside residing on the C6-O of the 2-deoxystreptamine was observed to undergo significant decomposition at the C2-C3 and O-C1 bonds. The comprehension of the CAD of known aminoglycosides aided in the identification of an unknown impurity in tobramycin. The unknown compound was initially detected by reverse phase high-performance liquid chromatography following dinitrofluorobenzene derivatization of the amino groups. The molecular weight of the dinitrobenzene derivative measured by LC mass spectrometry (MS) led to the detection of two isomeric impurities in tobramycin by LC-MS using an amino column. Their CAD spectra were subsequently acquired by LC-MS/MS. One of the two compounds was determined to be a known compound, 6"-O-carbamyltobramycin with the carbamyl group substituted on the glycoside residing on the C6-O of 2-deoxystreptamine. The fragmentation pattern of the other compound was interpreted as that the unknown was also a carbamyltobramycin. The carbamyl group was, however, substituted on 2-deoxystreptamine. It was speculated that the carbamyl group was substituted at the C1 amino group. This compound, to our knowledge, has not been reported before.

RNA molecules that specifically and stoichiometrically bind aminoglycoside antibiotics with high affinities.

RNA aptamers had previously been selected which were able to bind to the aminoglycoside antibiotic tobramycin with high affinity (Wang & Rando, 1995). Consensus sequences are found in a variety of constructs, and these sequences were mapped to stem-loop regions by Mfold secondary structure prediction. A tobramycin-based affinity cleavage reagent specifically cleaves the aptamers in their consensus regions. A fluorescence depolarization method is developed to accurately measure the affinity and stoichiometry of aminoglycoside binding to RNA constructs. An RNA aptamer (J6RNA) selected to bind to the aminoglycoside antibiotic tobramycin is shown to do so with an affinity of 0.77 nM and a stoichiometry of 1:1. (Fluorescently labeled) 5-carboxytetramethylrhodamine tobramycin (CRT) is used as a ligand in the fluorescence depolarization studies. J6RNA binding is quite specific for tobramycin, and weakly binds structurally related aminoglycosides with affinities 10(3)-10(4)-fold lower than that for tobramycin. Specific aminoglycoside binding aptamers of this type should be useful for revealing the rules of RNA-aminoglycoside recognition.

Synthesis of 5-deoxy-5-epifluoro derivatives of arbekacin, amikacin, and 1-N-[(S)-4-amino-2-hydroxybutanoyl]tobramycin (study on structure--toxicity relationships).

As part of a study on fluorination--toxicity relationships for aminoglycoside antibiotics, 5,3'-dideoxy-5-epifluorokanamycin B (10), 5,3',4'-trideoxy-5-epifluorokanamycin B (11), 1-N-[(S)-4-amino-2-hydroxybutanoyl]-5-deoxy-5-epifluorotobramyc in (19), 5-deoxy-5-epifluoroarbekacin (20), and 5-deoxy-5-epifluoroamikacin (21) have been prepared. The acute toxicities of these three 5-deoxy-5-epifluoro compounds showed values almost identical or similar to those for arbekacin (ABK) and amikacin (15), making a sharp contrast with the toxicities of the corresponding 5-deoxy-5-fluoro derivatives. This fact is explained on the basis of basicity changes (retention for the 5-epifluoro derivatives and reduction for the 5-fluoro derivatives) at the H2N-3 groups of the fluorinated compounds compared to the parent compounds; this hypothesis was substantiated by the pKa values at the H3N(+)-1, 3 groups (determined by the shift changes depending on pD values at C-2 and C-4, 6 in their 13C NMR spectra) of 2,5-dideoxy-5-epifluorostreptamine (23) and 2,5-dideoxy-5-fluorostreptamine (24), chosen as model compounds, and 2-deoxystreptamine (DST).

Specific binding of aminoglycoside antibiotics to RNA.

BACKGROUND: Aminoglycoside antibiotics interfere with ribosomal protein synthesis and with intron splicing. Various lines of evidence suggest that RNA is the molecular target for aminoglycosides, but little is known about the recognition process. Is recognition of a particular aminoglycoside specific for certain RNA structures? If so, what are the rules for recognition? We have begun to investigate this problem by in vitro selection of RNA molecules that can specifically bind to the aminoglycoside antibiotic tobramycin. RESULTS: An RNA diversity library was used to select for sequences capable of binding to the aminoglycoside antibiotic tobramycin. After six cycles of selection, 82% of the RNA bound to tobramycin specifically. The selected RNA was reverse-transcribed into DNA, which was then cloned. At low selection stringency, an extremely large number of clones, on the order of 10(7), produced RNAs capable of binding tobramycin with Kds in the microM range (values similar to that observed for the binding of tobramycin to Escherichia coli ribosomes). Sequencing of 18 of the clones revealed no obvious consensus sequence. At higher selection stringencies (Kds in the nM range) only two consensus sequences for binding were observed. CONCLUSIONS: We have shown that RNA molecules can be readily selected that bind the aminoglycoside tobramycin. The RNAs that bind tobramycin with high affinity contain consensus binding regions that may be confined to predicted stem-loop structures. These studies open the way for understanding the basis of RNA-aminoglycoside recognition.

Microscopic protonation constants in tobramycin. An NMR and pH study with the aid of partially N-acetylated derivatives.

Three tetra-N-acetyl derivatives and one tri-N-acetyl derivative of tobramycin (1) have been prepared by partial N-acetylation. Comparison of the pKa values, determined by NMR chemical shift titrations and pH titration of the derivatives, with those of unprotected 1 suggests that protonation equilibria at any particular amino group in 1 are not likely to be influenced by those at other sites. pH-Dependent conformational changes in 1 were assessed on the basis of 1H and 13C chemical shift changes in the derivatives.

1H, 13C, and 31P nuclear magnetic resonance spectral assignments for tobramycin, 2''-(adenosine-5'-phosphoryl)-tobramycin and 2''-(adenosine-5'-thiophosphoryl)-tobramycin.

Two enzymatically modified derivatives of tobramycin have been prepared by gentamicin nucleotidyl transferase-catalyzed adenylylation of tobramycin, using ATP and (Sp)-ATP alpha S as adenylylation substrates. (EC 2.7.7.46). The 1H, 13C, and 31P NMR spectra have been assigned for tobramycin, 2''-(adenosine-5'-phosphoryl)-tobramycin (TbAMP) and 2''-(adenosine-5'-thiophosphoryl)-tobramycin (TbAMPS). Several one- and two-dimensional NMR techniques have been utilized, notably, 1H-1H homonuclear correlation spectroscopy at 470 or 500 MHz and 13C-1H heteronuclear correlation spectroscopy at 50.3 MHz. The 1H assignments for tobramycin are similar to those previously reported for rings I and III of kanamycin A. The 13C assignments for tobramycin were similar to those previously reported, except for reversal of the assignments for anomeric carbons in the glycosyl rings. The 1H and 13C assignments for tobramycin were used to guide the assignments of the spectra for TbAMP and TbAMPS. Nearly complete assignments were obtained for these two derivatives of tobramycin. From the measured proton coupling constants, only small conformational changes were observed upon modification of tobramycin by adenylylation. From the proton and carbon spectra of the adenylylated derivatives the 2'' position is shown to be the site of adenylation. Large downfield shifts of the 2''proton and carbon resonances are easily observed and are more pronounced for TbAMPS than for TbAMP.

[Interaction of tobramycin and its derivatives with mitochondria in vitro]. / Interakcie tobramycínu a jeho derivátov s mitochondriami in vitro.

Tobramycin modified with fluorescamin yields fluorescent derivatives with a gradually changed structure and charge composition. Interactions of these drugs with mitochondria were investigated on the basis of the measurement of their inhibitory effect on the respiration (consumption of O2) of the isolated mitochondria. The results were compared with the effects of the original antibiotic agent. The use of the method of fluorescence polarization made it possible to determine the character of interactions. The procedure is a model experiment with possible wider use.

Selective quantitative determination of tobramycin from fermentation broth.

A derivative of Rhizobium meliloti 41 was constructed (strain GY654) which was resistant to apramycin and kanamycin but remained sensitive to tobramycin. Strain GY654 selectively measures tobramycin and 6"-O-carbamoyltobramycin in the presence of apramycin, kanamycin and 6"-O-carbamoylkanamycin B, therefore it is suitable for the rapid quantitation of 6"-O-carbamoyltobramycin and tobramycin in fermentation broths of Streptomyces tenebrarius and solvents containing antibiotic mixtures.

Photo-induced labelling of Escherichia coli ribosomes by a tobramycin analog.

An [3H]azidobenzyl derivative of tobramycin, a 4,6-disubstituted 2-deoxystreptamine aminoglycoside, has been synthesized, and its ability to label Escherichia coli 70-S ribosomes under photoactivation has been studied. Two concentrations of the photolabel, corresponding to the saturation of the two classes of tobramycin sites on the ribosomes, were used. The results show that, at high antibiotic concentrations which induce maximal misreading during protein synthesis, most of the ribosomal proteins are labelled. At low antibiotic concentration, which results in the saturation of the first-class sites, a few proteins of both subunits are labelled, including L6, S4, S5, and, to a lesser extent, L2, L13 and S18. The 30-S subunit is, on the whole, labelled more efficiently than the 50-S subunit.

[Quantitative determination of the antibiotic tobramycin using high-performance liquid chromatography]. / Kolichestvennoe opredelenie antibiotika tobramitsina s pomoshch'iu vyskooéffektivnoi zhidkostnoi khromatografii.

Separation of the main components of the nebramycin complex of apramycin, kanamycin B and tobramycin was achieved in the form of their 2,4-dinitrophenyl derivatives. A chromatograph SR 8000 of 'Spectra-Physics" and a column 'Sperisorb C6" (4.6 X 250 mm) with the granule size of 5 micrometers were used in the study with the mobile phase of acetone-trisbuffer, pH 7 in a ratio of 65 to 35, the flow rate of 1 ml/min, a temperature of 35 degrees C and detection at lambda 350 nm. Quantitative determination of the tobramycin purity level was performed with an equation developed for the given conditions. The purity levels of the reference tobramycin base and its sulfate were estimated. The results of the estimation corresponded to the data of biological titration.

Effects of N-alkylation and n-acylation on tobramycin activity.

The activities of tobramycin derivatives acetylated and ethylated on the 6'-N,2'-N and 3-N positions were examined. The MICs of these derivatives against tobramycin sensitive strains indicated that 2'-N-ethylated and 6'-N-ethylated derivatives have a fairly good activity, and confirmed that the 3-N position is the most important one for antibiotic activity since 3-N derivatives were less active. The MICs of these derivatives against tobramycin resistant strains, and their inactivation by tobramycin modifying enzymes were examined. These results showed that 2'-N or 6'-N ethylation protects the drug against inactivation by AAC(2') or AAC(6'), respectively, and 2'-N-ethyltobramycin and 6'-N-ethyltobramycin were active against strains containing these modifying enzymes. On the other hand, 3-N ethylation protects the drug against inactivation by AAC(3) but 3-N-ethyl tobramycin does not inhibit strains containing this enzyme.

Mechanism of action of aminoglycoside antibiotics. Binding studies of tobramycin and its 6'-N-acetyl derivative to the bacterial ribosome and its subunits.

6'-N-[14C]Acetyl-tobramycin and [3H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. THE 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6'-N-acetyl-tobramycin. The binding of [3H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6'-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6'-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6'-amino group.