Interaction between the antibiotic spiramycin and a ribosomal complex active in peptide bond formation

Dinos, George P.; Synetos, Dennis; Coutsogeorgopoulos, C.

doi:10.1021/bi00091a014

Cited by 24 publications

(32 citation statements)

References 36 publications

(36 reference statements)

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“…Inhibition of Peptide Bond Formation by Spiramycin-In agreement with previous results obtained at 10 mM Mg 2ϩ (16), spiramycin inactivates complex C at 6 mM Mg 2ϩ , almost irreversibly. Each time the plot reaches a plateau, which progressively bends down with increasing drug concentrations (plots not shown).…”

Section: Inhibition Of Peptide Bond Formation At High Concentrations supporting

confidence: 90%

“…Inactivation of Complex C by Spiramycin-The reaction of complex C with spiramycin in the absence of puromycin was examined as described previously (16).…”

Section: ) Of K Obs and [S] Values To Equationmentioning

confidence: 99%

“…Recently, it was discovered that 16-membered macrolides also exhibit an inhibitory effect on 50 S ribosomal subunit assembly (15). Nevertheless, spiramycin can inhibit peptide bond formation in most model cell-free systems by direct block-ing the PTase activity (11,16,17). This surprising effect seems to be due to the mycaminose/mycarose moiety of spiramycin, a disaccharide attached to the C5 position of the antibiotic (Fig.…”

mentioning

confidence: 99%

“…However, this concession cannot justify the binding of blasticidin S at two, nonoverlapping sites, nor can it explain the strengthening of spiramycin potency after preincubation of ribosomes with the drug (16). Experiments from more than 30 years ago have also indicated that the ionic environment is an essential factor that influences both the PTase activity and the interaction of antibiotics with ribosomes (18,19).…”

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confidence: 99%

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Polyamines Affect Diversely the Antibiotic Potency

Πετρόπουλος

Xaplanteri

Dinos

et al. 2004

Journal of Biological Chemistry

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The effects of spermine on peptidyltransferase inhibition by an aminohexosylcytosine nucleoside, blasticidin S, and by a macrolide, spiramycin, were investigated in 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. Kinetics revealed that blasticidin S, after a transient phase of interference with the A-site, is slowly accommodated near to the P-site so that peptide bond is still formed but with a lower catalytic rate constant. At high concentrations of blasticidin S (>10 ؋ K i ), a second drug molecule binds to a weaker binding site on ribosomes, and this may account for the onset of a subsequent mixed-noncompetitive inhibition phase. Spermine enhances the blasticidin S inhibitory effect by facilitating the drug accommodation to both sites. On the other hand, spiramycin (A) was found competing with puromycin for the A-site of AcPhe-tRNA⅐poly(U)⅐70 S ribosomal complex (C) via a two-step mechanism, according to which the fast formation of the encounter complex CA is followed by a slow isomerization to a tighter complex, termed C*A. In contrast to that observed with blasticidin S, spermine reduced spiramycin potency by decreasing the formation and stability of complex C*A. Polyamine effects on drug binding were more pronounced when a mixture of spermine and spermidine was used, instead of spermine alone. Our kinetic results correlate well with cross-linking and crystallographic data and suggest that polyamines bound at the vicinity of the antibiotic binding pockets modulate diversely the interaction of these drugs with ribosomes.Blasticidin S and spiramycin are representatives of two distinct antibiotic families, the aminohexosylcytosine nucleosides and the 16-membered lactone ring macrolides, respectively, both of which have been proposed to inhibit protein synthesis by binding to the large ribosomal subunit. Blasticidin S consists of a cytosine bonded to a pyranose ring, to which an amino acid-like substituent is attached (see Fig. 1). Therefore, it is not surprising that blasticidin S and puromycin have been considered as iso-structural, both with one another and with the 3Ј-end of aminoacyl-tRNA (1, 2). Consistently, blasticidin S has been found to inhibit the binding of CACCA (Phe) to the A-site of ribosomes (3) and to compete with designated A-site inhibitors (4). In addition, the inhibition of peptidyl-or AcPhe 1 -puromycin synthesis by this antibiotic shows a competitive phase in concert with noncompetitive or mixed-noncompetitive phases (5, 6), a fact supporting the notion that blasticidin S influences, at least transiently, the affinity of the A-site. Further support derives from chemical probing studies in Escherichia coli ribosomes (7), suggesting that blasticidin S protects A2439, one of the three nucleosides in domain V of 23 S rRNA, which show altered chemical reactivity on removal of the aminoacyl group from A-site-bound tRNAs (8). Moreover, A2439 is one of the preferable cross-linking...

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Section: Inhibition Of Peptide Bond Formation At High Concentrations supporting

confidence: 90%

“…Inactivation of Complex C by Spiramycin-The reaction of complex C with spiramycin in the absence of puromycin was examined as described previously (16).…”

Section: ) Of K Obs and [S] Values To Equationmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Polyamines Affect Diversely the Antibiotic Potency

Πετρόπουλος

Xaplanteri

Dinos

et al. 2004

Journal of Biological Chemistry

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“…When k Ϫ1 Ͼ Ͼ k 12 , the first binding step equilibrates much faster than the subsequent strong binding of the drug. Elimination of the fast variable (25) can then be used to simplify the slow binding step according to Reaction 2 (26).…”

Section: Quantitative Analysismentioning

confidence: 99%

Kinetics of Macrolide Action

Lovmar

Tenson

Ehrenberg

2004

Journal of Biological Chemistry

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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.

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Insights into functional modulation of catalytic RNA activity

et al. 2008

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SummaryRNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

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Interaction between the antibiotic spiramycin and a ribosomal complex active in peptide bond formation

Cited by 24 publications

References 36 publications

Polyamines Affect Diversely the Antibiotic Potency

Polyamines Affect Diversely the Antibiotic Potency

Kinetics of Macrolide Action

Insights into functional modulation of catalytic RNA activity

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