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 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.

Synthesis and Structures of Halo-Substituted Aroylhydrazones with Antimicrobial Activity

2012 ◽  
Vol 65 (4) ◽  
pp. 343 ◽  
Author(s):  
Mei Zhang ◽  
Dong-Mei Xian ◽  
Hai-Hua Li ◽  
Ji-Cai Zhang ◽  
Zhong-Lu You
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Inhibitory Concentration ◽  
Antimicrobial Activities ◽  
X Ray Diffraction ◽  
X Ray ◽  
E Coli ◽  
Diphenyl Tetrazolium Bromide

A series of new halo-substituted aroylhydrazones have been prepared and structurally characterized by elemental analysis, 1H NMR, 13C NMR, and IR spectra, and single crystal X-ray diffraction. The compounds were evaluated for their antibacterial (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescence) and antifungal (Candida albicans and Aspergillus niger) activities by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. Among the tested compounds, N′-(2-chloro-5-nitrobenzylidene)-2-fluorobenzohydrazide showed the most effective antimicrobial activity with minimum inhibitory concentration values of 0.82, 2.5, 1.7, 15.2, and 37.5 μg mL–1 against B. subtilis, S. aureus, E. coli, P. fluorescence, and C. albicans, respectively. The biological assay indicated that the presence of the electron-withdrawing groups in the aroylhydrazones improved their antimicrobial activities.

scholarly journals Insights into the Mode of Action of Novel Fluoroketolides, Potent Inhibitors of Bacterial Protein Synthesis

2013 ◽  
Vol 58 (1) ◽  
pp. 472-480 ◽  
Author(s):  
Marios G. Krokidis ◽  
Viter Márquez ◽  
Daniel N. Wilson ◽  
Dimitrios L. Kalpaxis ◽  
George P. Dinos
Keyword(s):  
Antimicrobial Agents ◽  
Lactone Ring ◽  
23S Rrna ◽  
Side Chains ◽  
Alkyl Aryl ◽  
Content Type ◽  
Future Design ◽  
Drug Concentrations ◽  
Lower Drug ◽  
Bacterial Protein Synthesis

ABSTRACTKetolides, the third generation of expanded-spectrum macrolides, have in the last years become a successful weapon in the endless war against macrolide-resistant pathogens. Ketolides are semisynthetic derivatives of the naturally produced macrolide erythromycin, displaying not only improved activity against some erythromycin-resistant strains but also increased bactericidal activity as well as inhibitory effects at lower drug concentrations. In this study, we present a series of novel ketolides carrying alkyl-aryl side chains at the C-6 position of the lactone ring and, additionally, one or two fluorine atoms attached either directly to the lactone ring at the C-2 position or indirectly via the C-13 position. According to our genetic and biochemical studies, these novel ketolides occupy the known macrolide binding site at the entrance of the ribosomal tunnel and exhibit lower MIC values against wild-type or mutant strains than erythromycin. In most cases, the ketolides display activities comparable to or better than the clinically used ketolide telithromycin. Chemical protection experiments usingEscherichia coliribosomes bearing U2609C or U754A mutations in 23S rRNA suggest that the alkyl-aryl side chain establishes an interaction with the U2609-A752 base pair, analogous to that observed with telithromycin but unlike the interactions formed by cethromycin. These findings reemphasize the versatility of the alkyl-aryl side chains with respect to species specificity, which will be important for future design of improved antimicrobial agents.

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 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 A polymerisation-associated conformational switch in FtsZ that enables treadmilling

2016 ◽  
Author(s):  
James M. Wagstaff ◽  
Matthew Tsim ◽  
María A. Oliva ◽  
Alba García-Sanchez ◽  
Danguole Kureisaite-Ciziene ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Division ◽  
Bacterial Cell Division ◽  
Conformational Switch ◽  
Septal Wall ◽  
Crystal Forms ◽  
X Ray ◽  
E Coli ◽  
X Ray Crystallography ◽  
Filament Assembly

AbstractBacterial cell division in many organisms involves a constricting cytokinetic ring that is orchestrated by the tubulin-like protein FtsZ. FtsZ forms dynamic filaments close to the membrane at the site of division that have recently been shown to treadmill around the division ring, guiding septal wall synthesis.Here, using X-ray crystallography ofStaphylococcus aureusSaFtsZ we reveal how an FtsZ can adopt two functionally distinct conformations, open and closed. The open form is found in SaFtsZ filaments formed in crystals and also in soluble filaments ofE. coliFtsZ as deduced by cryoEM. The closed form is found within several crystal forms of two non-polymerising SaFtsZ mutants and corresponds to many previous FtsZ structures from other organisms.We argue that FtsZ undergoes a polymerisation-associated conformational switch. We show that such a switch provides explanations for both how treadmilling may occur within a single-stranded filament, and why filament assembly is cooperative.

scholarly journals The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics

2006 ◽  
Vol 50 (7) ◽  
pp. 2500-2505 ◽  
Author(s):  
Katherine S. Long ◽  
Jacob Poehlsgaard ◽  
Corinna Kehrenberg ◽  
Stefan Schwarz ◽  
Birte Vester
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Drug Binding ◽  
Animal Origin ◽  
23S Rrna ◽  
Peptidyl Transferase ◽  
E Coli ◽  
Peptidyl Transferase Center ◽  
Drug Classes

ABSTRACT A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.

scholarly journals Size-Controlled Facile Synthesis of Silver Nanoparticles by Chemical Reduction Method and Their Antibacterial Performance Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Sanjoy Halder ◽  
Aninda Nafis Ahmed ◽  
M A Gafur ◽  
Gimyeong Seong ◽  
Muhammad Zamir Hossain
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Silver Nanoparticles ◽  
Chemical Reduction ◽  
Facile Synthesis ◽  
Capping Agents ◽  
X Ray ◽  
E Coli ◽  
Antibacterial Performance ◽  
Size Controlled

Abstract This article describes the size-controlled one-pot facile synthesis of silver nanoparticles (AgNPs) and their antibacterial response against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria. AgNPs coated with and without trisodium citrate (TSC) were synthesized using silver nitrate aqueous precursor and hydrazine as a reducing agent. The appearance of yellowish color with surface plasmon resonance (SPR) with maximum absorbance (λmax) at 416 nm from the ultraviolet-visible (UV–vis) spectrum of the product indicated primarily the formation of the desired product. X-ray diffraction (XRD) pattern confirmed the crystallinity of those products. Field emission scanning microscopy (FESEM) and transmission electron microscopy (TEM) images indicated only spherical AgNPs formation. The existence of a functional group of chemically bonded surface capping agents is confirmed by Fourier transform infrared (FTIR) and the themogravimetric analysis (TGA) weight loss assessment proved the attachment of good amount of capping agents. Energy dispersive X-ray (EDX) spectroscopic analysis of products revealed the existence of high percentage (96.36%) of silver. Exposing the synthesized AgNPs to Gram-positive S. aureus and Gram-negative E. coli bacteria, the zone inhibition was found to be 8 mm and 6 mm at a concentration of 50 mgL− 1, respectively. These results imply that TSC capped AgNPs can be considered as effective human pathogens for S. aureus and E. coli which is very inspiring.

scholarly journals Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alina Ornik-Cha ◽  
Julia Wilhelm ◽  
Jessica Kobylka ◽  
Hanno Sjuts ◽  
Attilio V. Vargiu ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Binding Pocket ◽  
Drug Binding ◽  
Side Chain ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Antibiotic Compounds ◽  
Periplasmic Domain ◽  
Transport Channels

AbstractUpon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.

scholarly journals Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

2015 ◽  
Vol 112 (29) ◽  
pp. 9004-9009 ◽  
Author(s):  
Hemant Kumar ◽  
Janet S. Finer-Moore ◽  
H. Ronald Kaback ◽  
Robert M. Stroud
Keyword(s):  
Hydrophobic Interactions ◽  
Side Chain ◽  
Lactose Permease ◽  
Protonation Site ◽  
Conformationally Constrained ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Biochemical Studies ◽  
Trp Mutant

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.

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