Polyamines Affect Diversely the Antibiotic Potency

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The effect of spermine on the inhibition of peptide-bond formation by clindamycin, an antibiotic of the Macrolide-Lincosamide-Streptogramins B family, was investigated in a cell-free system derived from Escherichia coli. In this system peptide bond is formed between puromycin, a pseudo-substrate of the A-site, and acetylphenylalanyl-tRNA, bound at the P-site of poly(U)-programmed 70 S ribosomes. Biphasic kinetics revealed that one molecule of clindamycin, after a transient interference with the A-site of ribosomes, is slowly accommodated near the P-site and perturbs the 70 S/acetylphenylalanyl-tRNA complex so that a peptide bond is still formed but with a lower velocity compared with that observed in the absence of the drug. The above mechanism requires a high temperature (25°C as opposed to 5°C). If this is not met, the inhibition is simple competitive. It was found that at 25°C spermine favors the clindamycin binding to ribosomes; the affinity of clindamycin for the A-site becomes 5 times higher, whereas the overall inhibition constant undergoes a 3-fold decrease. Similar results were obtained when ribosomes labeled with N 1 -azidobenzamidinospermine, a photo-reactive analogue of spermine, were used or when a mixture of spermine and spermidine was added in the reaction mixture instead of spermine alone. Polyamines cannot compensate for the loss of biphasic kinetics at 5°C nor can they stimulate the clindamycin binding to ribosomes. Our kinetic results correlate well with photoaffinity labeling data, suggesting that at 25°C polyamines bound at the vicinity of the drug binding pocket affect the tertiary structure of ribosomes and influence their interaction with clindamycin.Lincomycin and clindamycin ( Fig. 1) are lincosamides, still used as therapeutic agents in human diseases and some animal infections. Clindamycin, a semisynthetic derivative of lincomycin (7(S)-chloro-7-deoxylincomycin), is usually more active than the parent compound in the treatment of bacterial infections, in particular those caused by anaerobic species (1).Lincosamides act on the large ribosomal subunit and share an overlapping binding site with macrolides, streptogramins B, chloramphenicol, and other antibiotics affecting the PTase 2 center, as deduced from competition experiments and crossresistance data (for review, see Ref.2) as well as from twodimensional transferred nuclear Overhauser effect spectroscopy analysis (3). In agreement with most of these observations, chemical footprinting analysis (4, 5) and crystallographic studies (6, 7) have revealed that lincosamides interact with both the A-site and the P-site on the 50 S ribosomal subunit and would be expected to hamper the positioning of aminoacyl moieties of tRNAs at both sites. This last view is consistent with earlier binding studies investigating the competition for binding to ribosomes between lincomycin and 3Ј-terminus pentanucleotide fragments from N-AcLeu-tRNA Leu , Leu-tRNA Leu , and Phe-tRNA Phe (8, 9) as well as with the finding that lincosamides stimulate oligopeptidy...

Section: Inhibition Of Acphe-puromycin Synthesis By Clindamycin-supporting

confidence: 82%

Section: Target Moleculesupporting

confidence: 52%

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Unraveling New Features of Clindamycin Interaction with Functional Ribosomes and Dependence of the Drug Potency on Polyamines

Kouvela

Πετρόπουλος

Kalpaxis

2006

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“…Slow-binding enzyme inhibitors have been extensively discussed by Morrison and Walsh (35), and it has been suggested that such reactions should occur in two steps (36). In line with this, it was found that a number of macrolides, including erythromycin, bind to ribosomes in two steps.…”

Section: Table IImentioning

confidence: 54%

Kinetics of Macrolide Action

Lovmar

Tenson

Ehrenberg

2004

Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of fulllength proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacteriotoxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyltRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.

“…RNA folding, inhibits the binding of spiramycin, another 16-membered lactone ring macrolide (33). This prompted us to re-examine the interaction of tylosin with complex C in the presence of spermine.…”

Section: Inactivation Of Complex C By Tylosin-in Agreement Withmentioning

confidence: 99%

Stepwise Binding of Tylosin and Erythromycin to Escherichia coli Ribosomes, Characterized by Kinetic and Footprinting Analysis

Πετρόπουλος

Kouvela

Dinos

et al. 2008

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Erythromycin and tylosin are 14-and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg 2؉ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg 2؉ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment.Erythromycin and tylosin (Fig.