scholarly journals Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

2009 ◽  
Vol 53 (4) ◽  
pp. 1411-1419 ◽  
Author(s):  
Ekaterini C. Kouvela ◽  
Dimitrios L. Kalpaxis ◽  
Daniel N. Wilson ◽  
George P. Dinos
Keyword(s):  
Antimicrobial Activity ◽  
Attachment Site ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Distinct Mode ◽  
Alkyl Aryl ◽  
Resistant Strains ◽  
Exit Tunnel ◽  
A Site

ABSTRACT 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.

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

scholarly journals Ketolide Antimicrobial Activity Persists after Disruption of Interactions with Domain II of 23S rRNA

2004 ◽  
Vol 48 (10) ◽  
pp. 3677-3683 ◽  
Author(s):  
Guy W. Novotny ◽  
Lene Jakobsen ◽  
Niels M. Andersen ◽  
Jacob Poehlsgaard ◽  
Stephen Douthwaite
Keyword(s):  
Antimicrobial Activity ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Alkyl Aryl ◽  
Interaction Sites ◽  
Enhanced Resistance ◽  
Low Levels ◽  
Domain V ◽  
Block Protein Synthesis ◽  
Do So

ABSTRACT Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity. All macrolides and ketolides bind to the 50S ribosomal subunit, where they come into contact with adenosine 2058 (A2058) within domain V of the 23S rRNA and block protein synthesis. An additional interaction at nucleotide A752 in the rRNA domain II is made via the synthetic carbamate-alkyl-aryl substituent in the ketolides HMR3647 (telithromycin) and HMR3004, and this interaction contributes to their improved activities. Only a few macrolides, including tylosin, come into contact with domain II of the rRNA and do so via interactions with nucleotides G748 and A752. We have disrupted these macrolide-ketolide interaction sites in the rRNA to assess their relative importance for binding. Base substitutions at A752 were shown to confer low levels of resistance to telithromycin but not to HMR3004, while deletion of A752 confers low levels of resistance to both ketolides. Mutations at position 748 confer no resistance. Substitution of guanine at A2058 gives rise to the MLSB (macrolide, lincosamide, and streptogramin B) phenotype, which confers resistance to all the drugs. However, resistance to ketolides was abolished when the mutation at position 2058 was combined with a mutation in domain II of the same rRNA. In contrast, the same dual mutations in rRNAs conferred enhanced resistance to tylosin. Our results show that the domain II interactions of telithromycin and HMR3004 differ from each other and from those of tylosin. The data provide no indication that mutations within domain II, either alone or in combination with an A2058 mutation, can confer significant levels of telithromycin resistance.

scholarly journals Drug Resistance Mechanisms ofMycoplasma pneumoniaeto Macrolide Antibiotics

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Xijie Liu ◽  
Yue Jiang ◽  
Xiaogeng Chen ◽  
Jing Li ◽  
Dawei Shi ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Mechanisms ◽  
23S Rrna ◽  
Macrolide Antibiotics ◽  
Rrna Gene ◽  
Site Mutation ◽  
23S Rrna Gene ◽  
Resistant Strains ◽  
Domain V ◽  
The 16S Rrna Gene

Throat swabs from children with suspectedMycoplasma pneumoniae(M. pneumoniae) infection were cultured for the presence ofM. pneumoniaeand its species specificity using the 16S rRNA gene. Seventy-sixM. pneumoniaestrains isolated from 580 swabs showed that 70 were erythromycin resistant with minimum inhibitory concentrations (MIC) around 32–512 mg/L. FiftyM. pneumoniaestrains (46 resistant, 4 sensitive) were tested for sensitivity to tetracycline, ciprofloxacin, and gentamicin. Tetracycline and ciprofloxacin had some effect, and gentamicin had an effect on the majority ofM. pneumoniaestrains. Domains II and V of the 23S rRNA gene and the ribosomal protein L4 and L22 genes, both of which are considered to be associated with macrolide resistance, were sequenced and the sequences were compared with the corresponding sequences in M129 registered with NCBI and the FH strain. The 70 resistant strains all showed a 2063 or 2064 site mutation in domain V of the 23S rRNA but no mutations in domain II. Site mutations of L4 or L22 can be observed in either resistant or sensitive strains, although it is not known whether this is associated with drug resistance.

scholarly journals Kinetics of drug–ribosome interactions defines the cidality of macrolide antibiotics

2017 ◽  
Vol 114 (52) ◽  
pp. 13673-13678 ◽  
Author(s):  
Maxim S. Svetlov ◽  
Nora Vázquez-Laslop ◽  
Alexander S. Mankin
Keyword(s):  
Ribosomal Subunit ◽  
Drug Binding ◽  
Side Chain ◽  
Alkyl Aryl ◽  
Cell Growth Arrest ◽  
Drug Binding Site ◽  
Exit Tunnel ◽  
Critical Components ◽  
Slow Dissociation ◽  
Kinetics Of

Antibiotics can cause dormancy (bacteriostasis) or induce death (cidality) of the targeted bacteria. The bactericidal capacity is one of the most important properties of antibacterial agents. However, the understanding of the fundamental differences in the mode of action of bacteriostatic or bactericidal antibiotics, especially those belonging to the same chemical class, is very rudimentary. Here, by examining the activity and binding properties of chemically distinct macrolide inhibitors of translation, we have identified a key difference in their interaction with the ribosome, which correlates with their ability to cause cell death. While bacteriostatic and bactericidal macrolides bind in the nascent peptide exit tunnel of the large ribosomal subunit with comparable affinities, the bactericidal antibiotics dissociate from the ribosome with significantly slower rates. The sluggish dissociation of bactericidal macrolides correlates with the presence in their structure of an extended alkyl-aryl side chain, which establishes idiosyncratic interactions with the ribosomal RNA. Mutations or chemical alterations of the rRNA nucleotides in the drug binding site can protect cells from macrolide-induced killing, even with inhibitor concentrations that significantly exceed those required for cell growth arrest. We propose that the increased translation downtime due to slow dissociation of the antibiotic may damage cells beyond the point where growth can be reinitiated upon the removal of the drug due to depletion of critical components of the gene-expression pathway.

scholarly journals Binding and Action of CEM-101, a New Fluoroketolide Antibiotic That Inhibits Protein Synthesis

2010 ◽  
Vol 54 (12) ◽  
pp. 4961-4970 ◽  
Author(s):  
Beatriz Llano-Sotelo ◽  
Jack Dunkle ◽  
Dorota Klepacki ◽  
Wen Zhang ◽  
Prabhavathi Fernandes ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Hydrophobic Interactions ◽  
Lactone Ring ◽  
Drug Binding ◽  
23S Rrna ◽  
Side Chain ◽  
Alkyl Aryl ◽  
X Ray ◽  
E Coli ◽  
Chemical Probing

ABSTRACT We characterized the mechanism of action and the drug-binding site of a novel ketolide, CEM-101, which belongs to the latest class of macrolide antibiotics. CEM-101 shows high affinity for the ribosomes of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The ketolide shows high selectivity in its inhibitory action and readily interferes with synthesis of a reporter protein in the bacterial but not eukaryotic cell-free translation system. Binding of CEM-101 to its ribosomal target site was characterized biochemically and by X-ray crystallography. The X-ray structure of CEM-101 in complex with the E. coli ribosome shows that the drug binds in the major macrolide site in the upper part of the ribosomal exit tunnel. The lactone ring of the drug forms hydrophobic interactions with the walls of the tunnel, the desosamine sugar projects toward the peptidyl transferase center and interacts with the A2058/A2509 cleft, and the extended alkyl-aryl arm of the drug is oriented down the tunnel and makes contact with a base pair formed by A752 and U2609 of the 23S rRNA. The position of the CEM-101 alkyl-aryl extended arm differs from that reported for the side chain of the ketolide telithromycin complexed with either bacterial (Deinococcus radiodurans) or archaeal (Haloarcula marismortui) large ribosomal subunits but closely matches the position of the side chain of telithromycin complexed to the E. coli ribosome. A difference in the chemical structure of the side chain of CEM-101 in comparison with the side chain of telithromycin and the presence of the fluorine atom at position 2 of the lactone ring likely account for the superior activity of CEM-101. The results of chemical probing suggest that the orientation of the CEM-101 extended side chain observed in the E. coli ribosome closely resembles its placement in Staphylococcus aureus ribosomes and thus likely accurately reflects interaction of CEM-101 with the ribosomes of the pathogenic bacterial targets of the drug. Chemical probing further demonstrated weak binding of CEM-101, but not of erythromycin, to the ribosome dimethylated at A2058 by the action of Erm methyltransferase.

scholarly journals In VitroAntimicrobial Activity of a Siderophore Cephalosporin, S-649266, against Enterobacteriaceae Clinical Isolates, Including Carbapenem-Resistant Strains

2015 ◽  
Vol 60 (2) ◽  
pp. 729-734 ◽  
Author(s):  
Naoki Kohira ◽  
Joshua West ◽  
Akinobu Ito ◽  
Tsukasa Ito-Horiyama ◽  
Rio Nakamura ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Side Chain ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Class B ◽  
Resistant Strains ◽  
Cephalosporin Antibiotic ◽  
Catechol Moiety

ABSTRACTS-649266 is a novel siderophore cephalosporin antibiotic with a catechol moiety on the 3-position side chain. Two sets of clinical isolate collections were used to evaluate the antimicrobial activity of S-649266 againstEnterobacteriaceae. These sets included 617 global isolates collected between 2009 and 2011 and 233 β-lactamase-identified isolates, including 47 KPC-, 49 NDM-, 12 VIM-, and 8 IMP-producers. The MIC90values of S-649266 against the first set ofEscherichia coli,Klebsiella pneumoniae,Serratia marcescens,Citrobacter freundii,Enterobacter aerogenes, andEnterobacter cloacaeisolates were all ≤1 μg/ml, and there were only 8 isolates (1.3%) among these 617 clinical isolates with MIC values of ≥8 μg/ml. In the second set, the MIC values of S-649266 were ≤4 μg/ml against 109 strains among 116 KPC-producing and class B (metallo) carbapenemase-producing strains. In addition, S-649266 showed MIC values of ≤2 μg/ml against each of the 13 strains that produced other types of carbapenemases such as SME, NMC, and OXA-48. The mechanisms of the decreased susceptibility of 7 class B carbapenemase-producing strains with MIC values of ≥16 μg/ml are uncertain. This is the first report to demonstrate that S-649266, a novel siderophore cephalosporin, has significant antimicrobial activity againstEnterobacteriaceae, including strains that produce carbapenemases such as KPC and NDM-1.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

scholarly journals Context-specific action of macrolide antibiotics on the eukaryotic ribosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maxim S. Svetlov ◽  
Timm O. Koller ◽  
Sezen Meydan ◽  
Vaishnavi Shankar ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Amino Acid Sequences ◽  
Macrolide Antibiotics ◽  
Single Mutation ◽  
Sequence Motifs ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
Cellular Translation ◽  
Distinct Sequence ◽  
Exit Tunnel ◽  
Context Specific

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

scholarly journals Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Taolan Zhao ◽  
Yan-Ming Chen ◽  
Yu Li ◽  
Jia Wang ◽  
Siyu Chen ◽  
...  
Keyword(s):  
Protein Quality ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
Cryo Electron Microscopy ◽  
Cotranslational Folding ◽  
A Cell ◽  
Exit Tunnel ◽  
Hidden Layer ◽  
A Site

Abstract Background The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. Results In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides. Conclusions Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

scholarly journals Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 444
Author(s):  
Motoharu Hirano ◽  
Chihiro Saito ◽  
Hidetomo Yokoo ◽  
Chihiro Goto ◽  
Ryuji Kawano ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Hemolytic Activity ◽  
Helical Structure ◽  
Antimicrobial Activities ◽  
Side Chain ◽  
Membrane Disruption ◽  
Amino Acid Residues ◽  
Electrophysiological Measurements ◽  
Cell Membrane Disruption

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

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