Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes

Menninger, John R.; Otto, D P

doi:10.1128/aac.21.5.811

Cited by 144 publications

(97 citation statements)

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“…This is consistent with the long standing view that spiramycin acts during the early stages of protein synthesis by blocking the nascent polypeptide exit tunnel (13), a process that may also induce destabilization and premature dissociation of peptidyl-tRNAs from the ribosome (14). Recently, it was discovered that 16-membered macrolides also exhibit an inhibitory effect on 50 S ribosomal subunit assembly (15).…”

supporting

confidence: 68%

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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supporting

confidence: 68%

Polyamines Affect Diversely the Antibiotic Potency

Πετρόπουλος

Xaplanteri

Dinos

et al. 2004

Journal of Biological Chemistry

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“…It has been found that 16-membered ring macrolides such as josamycin, but not 14-membered macrolides such as erythromycin, inhibit the peptidyltransferase (PT) reaction itself (4,5). It has also been shown that both these types of macrolides cause peptidyl-tRNA dropoff from the ribosome (8). However, the kinetic relations between PT inhibition and drop-off have never been described, and the present results fill that gap in our knowledge.…”

Section: Discussioncontrasting

confidence: 35%

“…Peptidyl-tRNA Drop-off-It is known that macrolide treatment causes peptidyl-tRNA drop-off from ribosomes both in vitro (6,7) and in vivo (8). However, it is not known whether macrolides enhance the rate constant for peptidyl-tRNA dropoff or whether this reaction is an indirect effect of macrolidedependent stalling of ribosomes.…”

Section: Resultsmentioning

confidence: 99%

“…Erythromycin, in contrast, allows synthesis of short peptides before nascent protein elongation is inhibited (5). Macrolides also induce drop-off of peptidyl-tRNAs from translating ribosomes both in vitro (6,7) and in vivo (8). The macrolide induced drop-off events may be toxic for bacterial cells because accumulation of peptidyl-tRNA in the cell depletes pools of free tRNA isoacceptors (9 -11).…”

mentioning

confidence: 99%

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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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“…Early studies have demonstrated that erythromycin inhibits bacterial protein synthesis by binding to the 50S ribosomal unit, which prevents elongation of the peptide chain (Pestka, 1977). Spiramycin (and probably all macrolides) act similarly (Menninger & Otto, 1982). Erythromycin does not bind to mammalian 80S ribosomes, and this accounts in -part for -its selective toxicity (Mao, Putterman & Wiegand, 1970).…”

Section: Discussionmentioning

confidence: 99%