scholarly journals Interaction of Avilamycin with Ribosomes and Resistance Caused by Mutations in 23S rRNA

2002 ◽  
Vol 46 (11) ◽  
pp. 3339-3342 ◽  
Author(s):  
Christine B. Kofoed ◽  
Birte Vester
Keyword(s):  
Binding Site ◽  
Initiation Factor ◽  
23S Rrna ◽  
Cross Resistance ◽  
Halobacterium Halobium ◽  
Growth Promoter ◽  
A Site ◽  
Domain V ◽  
Trna Binding ◽  
Selection Of

ABSTRACT The antibiotic growth promoter avilamycin inhibits protein synthesis by binding to bacterial ribosomes. Here the binding site is further characterized on Escherichia coli ribosomes. The drug interacts with domain V of 23S rRNA, giving a chemical footprint at nucleotides A2482 and A2534. Selection of avilamycin-resistant Halobacterium halobium cells revealed mutations in helix 89 of 23S rRNA. Furthermore, mutations in helices 89 and 91, which have previously been shown to confer resistance to evernimicin, give cross-resistance to avilamycin. These data place the binding site of avilamycin on 23S rRNA close to the elbow of A-site tRNA. It is inferred that avilamycin interacts with the ribosomes at the ribosomal A-site interfering with initiation factor IF2 and tRNA binding in a manner similar to evernimicin.

scholarly journals Oxazolidinone Antibiotics Target the P Site on Escherichiacoli Ribosomes

2002 ◽  
Vol 46 (4) ◽  
pp. 1080-1085 ◽  
Author(s):  
Hiroyuki Aoki ◽  
Lizhu Ke ◽  
Susan M. Poppe ◽  
Toni J. Poel ◽  
Elizabeth A. Weaver ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Wide Spectrum ◽  
Anaerobic Bacteria ◽  
Elongation Factor ◽  
Peptide Bond ◽  
23S Rrna ◽  
Peptide Bonds ◽  
A Site ◽  
Domain V ◽  
Trna Binding

ABSTRACT The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

scholarly journals Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

2017 ◽  
Author(s):  
Wei Lin ◽  
Kalyan Das ◽  
David Degen ◽  
Abhishek Mazumder ◽  
Diego Duchi ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Resonance Energy ◽  
Active Pharmaceutical Ingredient ◽  
Conformational State ◽  
Initiation Factor ◽  
Structural Basis ◽  
Cross Resistance ◽  
Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

scholarly journals Mutation in 23S rRNA Associated with Macrolide Resistance in Neisseria gonorrhoeae

2002 ◽  
Vol 46 (9) ◽  
pp. 3020-3025 ◽  
Author(s):  
Lai-King Ng ◽  
Irene Martin ◽  
Gary Liu ◽  
Louis Bryden
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Promoter Region ◽  
Efflux Pump ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Cross Resistance ◽  
E Coli ◽  
Single Base Pair ◽  
Resistant Strains ◽  
Domain V

ABSTRACT Fifty-six azithromycin-resistant (MICs, 2.0 to 4.0 μg/ml) Neisseria gonorrhoeae strains with cross-resistance to erythromycin (MICs, 2.0 to 64.0 μg/ml), isolated in Canada between 1997 and 1999, were characterized, and their mechanisms of azithromycin resistance were determined. Most (58.9%) of them belonged to auxotype-serotype class NR/IB-03, with a 2.6-mDa plasmid. Based on resistance to crystal violet (MICs ≥ 1 μg/ml), 96.4% of these macrolide-resistant strains appeared to have increased efflux. Nine of the eleven strains selected for further characterization were found to have a promoter region mtrR mutation, a single-base-pair (A) deletion in the 13-bp inverted repeat, which is believed to cause overexpression of the mtrCDE-encoded efflux pump. The two remaining macrolide-resistant strains (erythromycin MIC, 64.0 μg/ml; azithromycin MIC, 4.0 μg/ml), which did not have the mutation in the mtrR promoter region, were found to have a C2611T mutation (Escherichia coli numbering) in the peptidyltransferase loop in domain V of the 23S rRNA alleles. Although mutations in domain V of 23S rRNA alleles had been reported in other bacteria, including E. coli, Streptococcus pneumoniae, and Helicobacter pylori, this is the first observation of these mutations associated with macrolide resistance in N. gonorrhoeae.

scholarly journals The expression of antibiotic resistance methyltransferase correlates with mRNA stability independently of ribosome stalling

2016 ◽  
pp. AAC.01806-16 ◽  
Author(s):  
Ekaterina Dzyubak ◽  
Mee-Ngan F. Yap
Keyword(s):  
Leader Peptide ◽  
23S Rrna ◽  
Cross Resistance ◽  
Nonsense Mutations ◽  
Stem Loop ◽  
Translational Initiation ◽  
Ribosome Stalling ◽  
Mrna Structure ◽  
Bacterial Ribosome ◽  
A Site

Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression ofermis activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the co-transcribederm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes theermShine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here we show that nonsense mutations in theStaphylococcus aureusErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence and presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of theermBL-ermBtranscript, and macrolide appears to mitigate mRNA cleavage at a site immediately downstream of theermBLSD sequence. The stabilizing effect of antibiotics on mRNA is not limited toermBL-ermB; cationic antibiotics representing a ribosome stalling inducer and a non-inducer increase the half-life of specific transcripts. These data unveil a new layer ofermBregulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

scholarly journals The C-terminal Subdomain (IF2 C-2) Contains the Entire fMet-tRNA Binding Site of Initiation Factor IF2

2000 ◽  
Vol 275 (4) ◽  
pp. 2447-2454 ◽  
Author(s):  
Roberto Spurio ◽  
Letizia Brandi ◽  
Enrico Caserta ◽  
Cynthia L. Pon ◽  
Claudio O. Gualerzi ◽  
...  
Keyword(s):  
Binding Site ◽  
Initiation Factor ◽  
Trna Binding

scholarly journals Linezolid and Tiamulin Cross-Resistance in Staphylococcus aureus Mediated by Point Mutations in the Peptidyl Transferase Center

2008 ◽  
Vol 52 (5) ◽  
pp. 1737-1742 ◽  
Author(s):  
Keith Miller ◽  
Colin J. Dunsmore ◽  
Colin W. G. Fishwick ◽  
Ian Chopra
Keyword(s):  
Staphylococcus Aureus ◽  
Point Mutations ◽  
Single Copy ◽  
23S Rrna ◽  
Cross Resistance ◽  
Limiting Factor ◽  
Peptidyl Transferase ◽  
Inhibit Protein Synthesis ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPSA phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.

scholarly journals Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

2016 ◽  
Vol 113 (27) ◽  
pp. 7527-7532 ◽  
Author(s):  
Stefan Arenz ◽  
Manuel F. Juette ◽  
Michael Graf ◽  
Fabian Nguyen ◽  
Paul Huter ◽  
...  
Keyword(s):  
Single Molecule ◽  
Antimicrobial Agents ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Cross Resistance ◽  
Single Molecule Fret ◽  
Arginine Residues ◽  
Peptidyltransferase Center ◽  
A Site

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra Terminal Domain

2020 ◽  
Author(s):  
Luke Adams ◽  
Lorna E. Wilkinson-White ◽  
Menachem J. Gunzburg ◽  
Stephen J. Headey ◽  
Martin J. Scanlon ◽  
...  
Keyword(s):  
Binding Site ◽  
Systematic Approach ◽  
Structural Information ◽  
Peptide Binding ◽  
Chemical Diversity ◽  
Chemical Probes ◽  
Protein Targets ◽  
Structure Activity ◽  
Peptide Binding Site ◽  
Selection Of

The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity >30-fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes.<br>

Design of Inhibitors for Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Enzyme of Leishmania mexicana

2020 ◽  
Vol 16 (6) ◽  
pp. 784-795
Author(s):  
Krisnna M.A. Alves ◽  
Fábio José Bonfim Cardoso ◽  
Kathia M. Honorio ◽  
Fábio A. de Molfetta
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Binding Site ◽  
Point Of View ◽  
New Drugs ◽  
Leishmania Mexicana ◽  
Receptor Complexes ◽  
Starting Point ◽  
Dynamics Simulations ◽  
Selection Of

Background:: Leishmaniosis is a neglected tropical disease and glyceraldehyde 3- phosphate dehydrogenase (GAPDH) is a key enzyme in the design of new drugs to fight this disease. Objective:: The present study aimed to evaluate potential inhibitors of GAPDH enzyme found in Leishmania mexicana (L. mexicana). Methods: A search for novel antileishmanial molecules was carried out based on similarities from the pharmacophoric point of view related to the binding site of the crystallographic enzyme using the ZINCPharmer server. The molecules selected in this screening were subjected to molecular docking and molecular dynamics simulations. Results:: Consensual analysis of the docking energy values was performed, resulting in the selection of ten compounds. These ligand-receptor complexes were visually inspected in order to analyze the main interactions and subjected to toxicophoric evaluation, culminating in the selection of three compounds, which were subsequently submitted to molecular dynamics simulations. The docking results showed that the selected compounds interacted with GAPDH from L. mexicana, especially by hydrogen bonds with Cys166, Arg249, His194, Thr167, and Thr226. From the results obtained from molecular dynamics, it was observed that one of the loop regions, corresponding to the residues 195-222, can be related to the fitting of the substrate at the binding site, assisting in the positioning and the molecular recognition via residues responsible for the catalytic activity. Conclusion:: he use of molecular modeling techniques enabled the identification of promising compounds as inhibitors of the GAPDH enzyme from L. mexicana, and the results obtained here can serve as a starting point to design new and more effective compounds than those currently available.

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