Conjugation with polyamines enhances the antibacterial and anticancer activity of chloramphenicol.

Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine-CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N(8),N(8)-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3'-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3'-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself.

Synthesis and antimicrobial activity of chloramphenicol-polyamine conjugates.

A series of chloramphenicol (CAM) amides with polyamines (PAs), suitable for structure-activity relationship studies, were synthesized either by direct attachment of the PA chain on the 2-aminopropane-1,3-diol backbone of CAM, previously oxidized selectively at its primary hydroxyl group, or from chloramphenicol base (CLB) through acylation with succinic or phthalic anhydride and finally coupling with a PA. Conjugates 4 and 5, in which the CLB moiety was attached on N4 and N1 positions, respectively, of the N(8),N(8)-dibenzylated spermidine through the succinate linker, were the most potent antibacterial agents. Both conjugates were internalized into Escherichia coli cells by using the spermidine-preferential uptake system and caused decrease in protein and polyamine content of the cells. Noteworthy, conjugate 4 displayed comparable activity to CAM in MRSA or wild-type strains of Staphylococcus aureus and Escherichia coli, but superior activity in E. coli strains possessing ribosomal mutations or expressing the CAM acetyltransferase (cat) gene. Lead compounds, and in particular conjugate 4, have been therefore discovered during the course of the present work with clinical potential.

Insights into the mode of action of novel fluoroketolides, potent inhibitors of bacterial protein synthesis.

Ketolides, the third generation of expanded-spectrum macrolides, have in the last years become a successful weapon in the endless war against macrolide-resistant pathogens. Ketolides are semisynthetic derivatives of the naturally produced macrolide erythromycin, displaying not only improved activity against some erythromycin-resistant strains but also increased bactericidal activity as well as inhibitory effects at lower drug concentrations. In this study, we present a series of novel ketolides carrying alkyl-aryl side chains at the C-6 position of the lactone ring and, additionally, one or two fluorine atoms attached either directly to the lactone ring at the C-2 position or indirectly via the C-13 position. According to our genetic and biochemical studies, these novel ketolides occupy the known macrolide binding site at the entrance of the ribosomal tunnel and exhibit lower MIC values against wild-type or mutant strains than erythromycin. In most cases, the ketolides display activities comparable to or better than the clinically used ketolide telithromycin. Chemical protection experiments using Escherichia coli ribosomes bearing U2609C or U754A mutations in 23S rRNA suggest that the alkyl-aryl side chain establishes an interaction with the U2609-A752 base pair, analogous to that observed with telithromycin but unlike the interactions formed by cethromycin. These findings reemphasize the versatility of the alkyl-aryl side chains with respect to species specificity, which will be important for future design of improved antimicrobial agents.

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes.

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

Oxidative Damage of Mussels Living in Seawater Enriched with Trace Metals, from the Viewpoint of Proteins Expression and Modification.

The impact of metals bioaccumulation in marine organisms is a subject of intense investigation. This study was designed to determine the association between oxidative stress induced by seawater enriched with trace metals and protein synthesis using as a model the mussels Mytilus galloprovincialis. Mussels were exposed to 40 µg/L Cu, 30 µg/L Hg, or 100 µg/L Cd for 5 and 15 days, and the pollution effect was evaluated by measuring established oxidative biomarkers. The results showed damage on the protein synthesis machine integrity and specifically on translation factors and ribosomal proteins expression and modifications. The exposure of mussels to all metals caused oxidative damage that was milder in the cases of Cu and Hg and more pronounced for Cd. However, after prolonged exposure of mussels to Cd (15 days), the effects receded. These changes that perturb protein biosynthesis can serve as a great tool for elucidating the mechanisms of toxicity and could be integrated in biomonitoring programs.

Distinct mode of interaction of a novel ketolide antibiotic that displays enhanced antimicrobial activity.

Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin-resistant strains, while maintaining their activity against erythromycin-susceptible ones. In this study, we present a new ketolide, K-1325, that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies, K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild-type Escherichia coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the alkyl-aryl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increased affinity of U2609C mutant ribosomes for the drug and rationalizing the enhanced effectiveness of this new ketolide.

Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine.

5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N1-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA(Phe) at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent 'loosening' of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.

New Chloramphenicol Derivatives from the Viewpoint of Anticancer and Antimicrobial Activity.

Over the last years, we have been focused on chloramphenicol conjugates that combine in their structure chloramphenicol base with natural polyamines, spermine, spermidine and putrescine, and their modifications. Conjugate 3, with spermidine (SPD) as a natural polyamine linked to chloramphenicol base, showed the best antibacterial and anticancer properties. Using 3 as a prototype, we here explored the influence of the antibacterial and anticancer activity of additional benzyl groups on N1 amino moiety together with modifications of the alkyl length of the aminobutyl fragment of SPD. Our data demonstrate that the novel modifications did not further improve the antibacterial activity of the prototype. However, one of the novel conjugates (4) showed anticancer activity without affecting bacterial growth, thus emerging as a promising anticancer agent, with no adverse effects on bacterial microflora when taken orally.

The contribution of the zinc-finger motif to the function of Thermus thermophilus ribosomal protein S14.

In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis.

Translational responses of Mytilus galloprovincialis to environmental pollution: integrating the responses to oxidative stress and other biomarker responses into a general stress index.

Heavy metals are commonly associated with the generation of reactive oxygen species (ROS), which may cause oxidative damage to several cellular macromolecules and organelles. In an attempt to correlate biomarker responses to oxidative stress, caged mussels (Mytilus galloprovincialis) were exposed for 30 days in a relatively clean site and two areas (Stations 1 and 2) unevenly polluted by heavy metals in Gulf of Patras (Greece). Three periods of caging were: one in winter, the second in spring, and the third in autumn. Heavy metal content was determined in digestive glands of the exposed mussels as a measure of metal pollution, metallothionein content as an adaptive and detoxifying index, lysosomal membrane stability as a biomarker of general stress, superoxide radical production and lipid peroxidation as indicators of oxidative stress, and micronucleus frequency in gill cells as an index of chromosomal damage. Considering that protein-synthesizing machinery is one of the candidate targets for ROS, the in vivo activity of ribosomes in digestive glands was also tested. Compared with the reference samples, mussels transplanted to Station 1 showed increased levels of heavy metals and metallothionein in digestive glands, lower lysosomal membrane stability, higher values in oxidative stress indices, reduced activity of ribosomes, and increased chromosomal damage in gill cells. In addition, run-off ribosomes isolated from mussels transplanted to Station 1 were less efficient at initiating protein synthesis in a cell-free system than those from mussels in the reference site. Mussels transplanted to Station 2 exhibited similar but less pronounced responses. Statistical analysis revealed a strong positive correlation of ribosomal activity with lysosomal membrane stability, as well as a significant negative correlation with the oxidative stress indices, metallothionein content, micronucleus frequency, and the digestive gland content in Cr, Cu and Mn. Integration of all the measured biomarker responses into one general "stress index" demonstrated a clear distinction between the sampling sites, allowing classification along a pollution gradient (reference site

Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu.

The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase--the essential catalytic activity of the large ribosomal subunit--also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA*EF-Tu*GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit.

Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions.

Polyamine binding to 23S rRNA was investigated, using a photoaffinity labeling approach. This was based on the covalent binding of a photoreactive analog of spermine, N1-azidobenzamidino (ABA)-spermine, to Escherichia coli ribosomes or naked 23S rRNA under mild irradiation conditions. The cross-linking sites of ABA-spermine in 23S rRNA were determined by RNase H digestion and primer-extension analysis. Domains I, II, IV and V in naked 23S rRNA were identified as discrete regions of preferred cross-linking. When 50S ribosomal subunits were targeted, the interaction of the photoprobe with the above 23S rRNA domains was elevated, except for helix H38 in domain II whose susceptibility to cross-linking was greatly reduced. In addition, cross-linking sites were identified in domains III and VI. Association of 30S with 50S subunits, poly(U), tRNA(Phe) and AcPhe-tRNA to form a post-translocation complex further altered the cross-linking, in particular to helices H11-H13, H21, H63, H80, H84, H90 and H97. Poly(U)-programmed 70S ribosomes, reconstituted from photolabeled 50S subunits and untreated 30S subunits, bound AcPhe-tRNA in a similar fashion to native ribosomes. However, they exhibited higher reactivity toward puromycin and enhanced tRNA-translocation efficiency. These results suggest an essential role for polyamines in the structural and functional integrity of the large ribosomal subunit.

Oxidative damage of 18S and 5S ribosomal RNA in digestive gland of mussels exposed to trace metals.

Numerous studies have shown the ability of trace metals to accumulate in marine organisms and cause oxidative stress that leads to perturbations in many important intracellular processes, including protein synthesis. This study is mainly focused on the exploration of structural changes, like base modifications, scissions, and conformational changes, caused in 18S and 5S ribosomal RNA (rRNA) isolated from the mussel Mytilus galloprovincialis exposed to 40µg/L Cu, 30µg/L Hg, or 100µg/L Cd, for 5 or 15days. 18S rRNA and 5S rRNA are components of the small and large ribosomal subunit, respectively, found in complex with ribosomal proteins, translation factors and other auxiliary components (metal ions, toxins etc). 18S rRNA plays crucial roles in all stages of protein synthesis, while 5S rRNA serves as a master signal transducer between several functional regions of 28S rRNA. Therefore, structural changes in these ribosomal constituents could affect the basic functions of ribosomes and hence the normal metabolism of cells. Especially, 18S rRNA along with ribosomal proteins forms the decoding centre that ensures the correct codon-anticodon pairing. As exemplified by ELISA, primer extension analysis and DMS footprinting analysis, each metal caused oxidative damage to rRNA, depending on the nature of metal ion and the duration of exposure. Interestingly, exposure of mussels to Cu or Hg caused structural alterations in 5S rRNA, localized in paired regions and within loops A, B, C, and E, leading to a continuous progressive loss of the 5S RNA structural integrity. In contrast, structural impairments of 5S rRNA in mussels exposed to Cd were accumulating for the initial 5days, and then progressively decreased to almost the normal level by day 15, probably due to the parallel elevation of metallothionein content that depletes the pools of free Cd. Regions of interest in 18S rRNA, such as the decoding centre, sites implicated in the binding of tRNAs (A- and P-sites) or translation factors, and areas related to translation fidelity, were found to undergo significant metal-induced conformational alterations, leading either to loosening of their structure or to more compact folding. These modifications were associated with parallel alterations in the translation process at multiple levels, a fact suggesting that structural perturbations in ribosomes, caused by metals, pose significant hurdles in translational efficiency and fidelity.

Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers.

Protein synthesis down-regulation is a life-saving mechanism for many organisms exposed to xenobiotics that threaten normal life. The present study was designed to assess the spatial and seasonal variability of global protein synthesis, determined in the microsomal fraction of digestive glands from caged Mytilus galloprovincialis mussels exposed for 30 days in a relatively clean region and two unevenly polluted areas (Stations 1 and 2) along the Gulf of Patras (Greece). The in vivo activity of translating ribosomes was evaluated by analyzing the translating ribosomes, polysome content, which may serve as an indicator of the efficiency of the protein-synthesizing machinery. To correlate with classical biomonitoring strategies, various biomarkers were measured in digestive glands, including metallothionein content, heavy-metal content, and lysosomal membrane stability. In parallel, gill cells were examined for micronucleus frequency. Metal ion concentrations were also estimated in the surrounding waters as a measure of metal exposure. Substantially lower polysome content was recorded in caged mussels collected from Station 1, in particular during the winter and spring sampling. As verified by chemical analysis of the seawater and measurement of other biomarkers, Station 1 was more contaminated than Station 2. Polysome content was found negatively correlated with metallothionein levels, micronucleus frequency and cytosolic Cu and Hg in all seasons. In addition, negative correlations were obtained between polysome content and lysosomal membrane stability in winter and spring. A progressive increase in polysomes was observed from winter to autumn, in particular in samples from Station 1. A non-uniform trend was detected in 80S ribosomal monosomes, whereas the seasonal changes in ribosomal subunits were opposite to those found in polysome content. Comparisons between seasonal and local site-specific influences on polysome content provides evidence that winter and spring are the most appropriate sampling seasons for application of translation activity as a possible biomarker in biomonitoring studies.

The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions.

A photoreactive analogue of spermine, N1-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5' domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem-loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.

Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action.

Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3'-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg2+, 100 mM NH4+, 100 microM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA.poly(U).70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes.

Chloramphenicol Derivatives as Antibacterial and Anticancer Agents: Historic Problems and Current Solutions.

Chloramphenicol (CAM) is the D-threo isomer of a small molecule, consisting of a p-nitrobenzene ring connected to a dichloroacetyl tail through a 2-amino-1,3-propanediol moiety. CAM displays a broad-spectrum bacteriostatic activity by specifically inhibiting the bacterial protein synthesis. In certain but important cases, it also exhibits bactericidal activity, namely against the three most common causes of meningitis, Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis. Resistance to CAM has been frequently reported and ascribed to a variety of mechanisms. However, the most important concerns that limit its clinical utility relate to side effects such as neurotoxicity and hematologic disorders. In this review, we present previous and current efforts to synthesize CAM derivatives with improved pharmacological properties. In addition, we highlight potentially broader roles of these derivatives in investigating the plasticity of the ribosomal catalytic center, the main target of CAM.

On the structural and functional importance of the highly conserved Glu56 of Thermus thermophilus L4 ribosomal protein.

The structural and functional importance of the highly conserved amino acid residue glutamic acid 56 (Glu56) of the ribosomal protein L4 from Thermus thermophilus (TthL4) has been investigated by replacing this residue by alanine or glutamine, and by incorporating the resulted mutants into Escherichia coli ribosomes. The catalytic properties of peptidyltransferase estimated for the mutants as well as for the wild-type TthL4 by the puromycin reaction, were quite different. The binding of tRNA to the P and A-site was affected. In addition, replacement of the native L4 protein by wild-type TthL4 or by TthL4-Ala56 mutant resulted in reduced capability of 50S subunits for association with 30S subunits. In contrast, neither the assembly of the 50S subunits nor the fixation of the tRNA 3'-end at the P or A-site was affected. These results are used to discuss critically the hypothesis that the delta-carboxyl group of the highly conserved Glu56 is essential for stabilizing a flexible loop of L4, which extended into the ribosome interior region, influences the mechanism of peptide bond formation. Mutations concerning the semi-conserved glycine 55 (Gly55) were investigated. Replacement of Gly55 by serine did not affect the measured functions. In contrast, replacement of Gly55 by alanine resulted in enhanced peptidyltransferase activity and increased tRNA affinity for the P and A-sites, indicating a possible implication of this amino acid in the local loop conformation of TthL4.

Synthesis and evaluation of chloramphenicol homodimers: molecular target, antimicrobial activity, and toxicity against human cells.

As fight against antibiotic resistance must be strengthened, improving old drugs that have fallen in reduced clinical use because of toxic side effects and/or frequently reported resistance, like chloramphenicol (CAM), is of special interest. Chloramphenicol (CAM), a prototypical wide-spectrum antibiotic has been shown to obstruct protein synthesis via binding to the bacterial ribosome. In this study we sought to identify features intensifying the bacteriostatic action of CAM. Accordingly, we synthesized a series of CAM-dimers with various linker lengths and functionalities and compared their efficiency in inhibiting peptide-bond formation in an Escherichia coli cell-free system. Several CAM-dimers exhibited higher activity, when compared to CAM. The most potent of them, compound 5, containing two CAM bases conjugated via a dicarboxyl aromatic linker of six successive carbon-bonds, was found to simultaneously bind both the ribosomal catalytic center and the exit-tunnel, thus revealing a second, kinetically cryptic binding site for CAM. Compared to CAM, compound 5 exhibited comparable antibacterial activity against MRSA or wild-type strains of Staphylococcus aureus, Enterococcus faecium and E. coli, but intriguingly superior activity against some CAM-resistant E. coli and Pseudomonas aeruginosa strains. Furthermore, it was almost twice as active in inhibiting the growth of T-leukemic cells, without affecting the viability of normal human lymphocytes. The observed effects were rationalized by footprinting tests, crosslinking analysis, and MD-simulations.

Evaluation of the distribution of Paclitaxel by immunohistochemistry and nuclear magnetic resonance spectroscopy after the application of a drug-eluting balloon in the porcine ureter.

BACKGROUND AND PURPOSE: The urothelium represents "the tightest and most impermeable barrier in the body." We investigated the distribution of paclitaxel (PTX) in the ureteral wall after the inflation of a paclitaxel-eluting balloon (PEB) in an attempt to elucidate the possibility of clinical application of PEBs in the ureter. MATERIALS AND METHODS: Nine domestic pigs were used. Nine PEBs and nine conventional percutaneous angioplasty balloons (CB) were inflated in the right and left ureter of each animal, respectively. The ureter treated by CB was the control for the contralateral ureter. Specimens were removed: Immediately after inflation (group A), after 12 hours (group B), and after 24 hours (group C). Two samples were obtained from each ureter of groups A, B and C. One sample was investigated by nuclear magnetic resonance spectroscopy (NMR), the other by histology and immunohistochemistry (IHC) using a specific for PTX polyclonal antibody. RESULTS: Reduced inflammation was observed in the group B and C samples in comparison with their controls. PTX was distributed mostly in the urothelium and submucosal layer in group A (IHC). The agent was present in the urothelial, submucosal, and muscle layer in groups B and C. The concentration of PTX (NMR) has been reduced in group C compared with the tissue extracts of group B. CONCLUSION: The distribution of PTX includes the urothelial, submucosal, and smooth muscle layers. Inflammation was reduced in the case of drug-eluting balloons.