Effects of macrolides on peptide-bond formation and translocation

Mao, James C.-H.; Robishaw, Ellen E.

doi:10.1021/bi00787a014

Cited by 66 publications

(49 citation statements)

References 29 publications

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“…1C) (13). This is in line with results from biochemical experiments, showing that carbomycin A inactivates peptidyl transfer in an artificial system with puromycin as the acceptor and N-acetyl-Phe-tRNA Phe as the donor (4,5). In contrast, experiments in a more realistic system with heteropolymeric mRNAs show that both fMet-Val-tRNA Val and fMet-Gly-Ile-tRNA Ile can be formed on ribosomes in complex with josamycin (7).…”

Section: Resultssupporting

confidence: 76%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…All macrolides bind competitively to a site in the vicinity of the peptidyltransferase center (3), but direct inhibition of the peptidyltransferase reaction has been observed only for macrolides with 16-membered lactone rings (4,5). 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).…”

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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Section: Resultssupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetics of Macrolide Action

Lovmar

Tenson

Ehrenberg

2004

Journal of Biological Chemistry

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“…Previous work indicates that chloramphenicol and certain macrolides fail to inhibit poly(Phe) synthesis in cell extracts (25,32,34). We initially tested azithromycin and chloramphenicol in the A/T system, and a Ͻ5% decrease in poly(Phe) synthesis was detected.…”

Section: Discussionmentioning

confidence: 99%

Discovery and Analysis of 4 H -Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors

Ribble

Hill

Ochsner

et al. 2010

Antimicrob Agents Chemother

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Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyltRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC 50 s) ranging from 3 to 14 M. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five-or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 g/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

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“…An example is carbomycin A, a 16-membered macrolide that carries a large mycaminose-mycarose sugar moiety. When bound to a H. marismortui ribosome (19), this extension reaches the peptidyl-transferase center, a finding correlating well with dissociation of peptidyl-tRNAs containing 2-4 residues (19,65,75,91). In contrast, erythromycin, which binds to the macrolide pocket but does not possess a mycaminose-mycarose sugar, causes dissociation of peptidyl-tRNAs containing 6-8 residues (65).…”

Section: Troleandomycin: a Macrolide Interacts With Two Domains And Tmentioning

confidence: 99%

ANTIBIOTICS TARGETING RIBOSOMES: Resistance, Selectivity, Synergism, and Cellular Regulation

Yonath

2005

Annu. Rev. Biochem.

205

145

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Key Words ribosomal crystallography, peptide-bond formation, antibiotic synergism, nascent protein exit tunnel ■ Abstract Antibiotics target ribosomes at distinct locations within functionally relevant sites. They exert their inhibitory action by diverse modes, including competing with substrate binding, interfering with ribosomal dynamics, minimizing ribosomal mobility, facilitating miscoding, hampering the progression of the mRNA chain, and blocking the nascent protein exit tunnel. Although the ribosomes are highly conserved organelles, they possess subtle sequence and/or conformational variations. These enable drug selectivity, thus facilitating clinical usage. The structural implications of these differences were deciphered by comparisons of high-resolution structures of complexes of antibiotics with ribosomal particles from eubacteria resembling pathogens and from an archaeon that shares properties with eukaryotes. The various antibiotic-binding modes detected in these structures demonstrate that members of antibiotic families possessing common chemical elements with minute differences might bind to ribosomal pockets in significantly different modes, governed by their chemical properties.

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Effects of macrolides on peptide-bond formation and translocation

Abstract: adenosine; GMPPCP, 5'-guanylylmethylenediphosphonate; 1 optical density unit is the amount of material which in 1.0 ml would yield a value of 1.0 for the optical density measured at 260 µ in a cuvet with a path length of 1.0cm.

Cited by 66 publications

References 29 publications

Kinetics of Macrolide Action

Kinetics of Macrolide Action

Discovery and Analysis of 4 H -Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors

ANTIBIOTICS TARGETING RIBOSOMES: Resistance, Selectivity, Synergism, and Cellular Regulation

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