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Aminoglycoside antibiotics induce translational misreading. (A–D) Chemical structures of aminogylcoside antibiotics (A) paromomycin , (B) neomycin, (C) gentamycin and (D) kanamycin. (E) Overview of the streptomycin (Stp, red) and aminoglycoside paromomycin (Par, blue) binding site on the 30S subunit. Helix 44 (h44, yellow), ribosomal protein S12 (light green) and the relative positions of mRNA (orange), A-(teal), P-(purple) and E-tRNA (cyan) are shown for reference. (F–I) Conformation of A1492 and A1493 in h44 in (F) the native 30S subunit, (G) the presence of mRNA (59-UUU codon in A site) and cognate A-tRNA (anticodon 59-GGA), (H) the presence of mRNA (59-UUU codon in A site) and near-cognate A-tRNA (anticodon 59-GAA), (I) the presence of paromomycin (Par, blue), mRNA (59-UUU codon in A site) and near-cognate A-tRNA (anticodon 59-GAA). Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by Carol Pfeffer
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Protein synthesis is one of the major targets in the cell for antibiotics. This review endeavors to provide a comprehensive "post-ribosome structure" A-Z of the huge diversity of antibiotics that target the bacterial translation apparatus, with an emphasis on correlating the vast wealth of biochemical data with more recently available ribosome stru...
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... that target the ribosome contain a 2-deoxystreptamine (2-DOS/ring II) group with a distinct patterns of sugar substitutions depending on the mem- ber. For example, the paromomycins and neomycins contain sugars at the C4 and C5 positions (Figures 7A, 7B), whereas gentamycin and kanamycin are 4,6 di- substituted ( Figures 7C, 7D). The 4,6 di-substituted aminoglycosides are clinically preferred, with the pre- dominant examples being gentamycin (Garamycin ® introduced in the mid 1960s by Schering-Plough) and two kanamycin derivatives, tobramycin (Nebcin ® mar- keted Eli Lilly and Company) and amikacin (marketed under the name Amikin ® by Bristol-Myers Squibb). ...
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... 4,6 di-substituted aminoglycosides are clinically preferred, with the pre- dominant examples being gentamycin (Garamycin ® introduced in the mid 1960s by Schering-Plough) and two kanamycin derivatives, tobramycin (Nebcin ® mar- keted Eli Lilly and Company) and amikacin (marketed under the name Amikin ® by Bristol-Myers Squibb). All aminoglycosides bind within an internal loop in h44 of the 30S subunit, which comprises the decoding site ( Figure 7E) (reviewed by Ogle et al., 2003). This has been observed biochemically by chemical probing (Moazed and Noller, 1987b;Woodcock et al., 1991) as well as structurally in complexes of aminoglycosides bound to small RNA fragments mimicking h44 (Fourmy et al., , yellow), ribosomal protein S12 (light green) and the relative positions of mRNA (orange), A-(teal), P-(purple) and E-tRNA (cyan) are shown for reference. ...
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... decoding, the ribosome monitors the codon- anticodon interaction to ensure that the A-tRNA is cognate to the mRNA. This monitoring involves two universally conserved nucleotides of the 16S rRNA, A1492 and A1493 ( Figure 7F), which flip-out of helix 44 in the 30S subunit to analyze the minor groove of the codon-anticodon duplex ( Figure 7G) (Ogle et al., 2001). Presumably the energy required to flip-out A1492 and A1493 during decoding is compensated for by additional A-minor interactions established with the codon-anticodon duplex, thus stabilizing this "flipped- out" conformation ( Ogle et al., 2001). ...
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... decoding, the ribosome monitors the codon- anticodon interaction to ensure that the A-tRNA is cognate to the mRNA. This monitoring involves two universally conserved nucleotides of the 16S rRNA, A1492 and A1493 ( Figure 7F), which flip-out of helix 44 in the 30S subunit to analyze the minor groove of the codon-anticodon duplex ( Figure 7G) (Ogle et al., 2001). Presumably the energy required to flip-out A1492 and A1493 during decoding is compensated for by additional A-minor interactions established with the codon-anticodon duplex, thus stabilizing this "flipped- out" conformation ( Ogle et al., 2001). ...
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... the energy required to flip-out A1492 and A1493 during decoding is compensated for by additional A-minor interactions established with the codon-anticodon duplex, thus stabilizing this "flipped- out" conformation ( Ogle et al., 2001). In the presence of near-cognate tRNA these compensatory interactions are obviously insufficient to stabilize the flipped out A1492 and A1493 and thus the near-cognate tRNA dissociates ( Figure 7H). However, in the presence of paromomycin (Par), the uncompensated loss of energy is absorbed by Par since the drug has already induced A1492 and A1493 to flip-out and stabilized them in this open conforma- tion ( Figure 7I). ...
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... the presence of near-cognate tRNA these compensatory interactions are obviously insufficient to stabilize the flipped out A1492 and A1493 and thus the near-cognate tRNA dissociates ( Figure 7H). However, in the presence of paromomycin (Par), the uncompensated loss of energy is absorbed by Par since the drug has already induced A1492 and A1493 to flip-out and stabilized them in this open conforma- tion ( Figure 7I). The outcome being that a near-cognate tRNA becomes fully accommodated into the A-site, which results in mis-incorporation of an amino acid (reviewed by Ogle et al., 2003). ...
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... B (Hyg B) is an atypical aminoglyco- side produced by Streptomyces hygroscopicus that is structurally and functionally unique compared to other aminoglycosides. Although Hyg B also binds in h44 of the 30S subunit, the location is slightly displaced towards the top of h44 when compared to the position of, for exam- ple, Par ( Figure 7E) Borovinskaya et al., 2008). Hyg B has only a modest effect on transla- tional fidelity, consistent with the observation that bind- ing of Hyg B does not induce the same conformational changes in A1492 and A1493 as seen for Par ( Brodersen et al., 2000;Borovinskaya et al., 2008). ...
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... Par, Hyg B is a powerful translocation inhibitor (Cabanas et al., 1978;Hausner et al., 1988;Peske et al., 2004), but unlike Par, Hyg B also inhibits back-translocation (Borovinskaya et al., 2008). Hyg B directly contacts the mRNA in the P-site ( Figure 7E) and places A1493 in a position to inter- act with the A-site codon, suggesting that Hyg B inhibits translocation through confinement of the mRNA in the A-and P-sites (Borovinskaya et al., 2008). ...
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... induces translational misreading (Kurland et al., 1996). Stp binds to a distinct site on the ribosome ( Figure 7E) and therefore mediates its inhibitory and misreading effects by an unrelated mechanism to aminoglycosides. Unlike aminoglycosides that bind in h44, Stp has a single bind- ing site on the 30S subunit that connects helices from all four different domains of the 16S rRNA, namely h1, h18, h27 and h44, and makes interactions with r-protein S12 ( Figure 8B). ...
Similar publications
Growing evidence suggests that many ribosome-targeting antibiotics inhibit protein synthesis context specifically, which has important implications for drug development. New work reveals the structural basis of context-specific action of the classic translation inhibitor chloramphenicol and the oxazolidinones linezolid and radezolid.
Citations
... [34] In the case of controls like Linezolid; biochemical and structural evidence previously reviewed, indicates that Linezolid binds to the peptidyl transferase center of the large ribosomal subunit (50s), in a position that overlaps with the aminoacyl residue of an A site attached to tRNA. [35,36] Based on our study, the binding site of the nine N-alkyl substituted morpholine compounds and Linezolid should prevent the correct placement of the aminoacyl residue of the A-tRNA and inhibit the formation of peptide bonds. ...
In this work, we synthesized a series of nine tetraalkylammonium salts derived from N‐methyl morpholine and assessed their antibacterial efficacy against Methicillin‐Resistant Staphylococcus aureus (MRSA). The investigated morpholinium cations differ by the length of one linear alkyl chain, which ranges from 1 to 18 carbon atoms. We found that compounds with alkyl chains in the n‐C12H25‐(n‐dodecyl) to C16H33‐(n‐hexadecyl) display the highest bactericidal effects, exhibiting Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of 3.9 μg/mL – surpassing the effectiveness of the commercial drug Linezolid. Conversely, compounds with shorter chains (<5 carbon atoms) are inactive against MRSA, establishing a clear structure‐activity relationship. Assays on A. salina reveal that short‐alkyl‐chain morpholinium derivatives, inactive against MRSA), are moderately toxic, while the strongest bactericides of our set demonstrate high or extreme toxicity to A. salina. Our findings underscore the potential of morpholine derivatives as a means to control Methicillin‐Resistant Staphylococcus aureus and pinpoint the need to develop safe applications for these compounds given their potential toxicity.
... To probe the basis of TET insensitivity to nitrate and rule out TET-specific effects, we tested several other translation inhibitors with different features (Fig 6). TET acts on the 30S subunit of the ribosome by blocking binding of charged tRNAs to the A site and inhibits translation initiation [68,69]. Chloramphenicol (CHL) targets the 50S subunit at the peptidyl transferase Biofilm assays were performed using peg lids in liquid 50:50 media with or without 50 mM potassium nitrate. ...
... Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics center and inhibits peptide bond formation [69]. Biofilm stimulation in response to CHL was not impacted by nitrate addition, suggesting that lack of nitrate suppression is not specific to 30S targeting antibiotics. ...
... The biofilm response to AZI and ERM was unaffected by nitrate addition, indicating that nitrate insensitivity is likely not determined by context-specific inhibition, leaky translation, or charge-dependent uptake. We tested spectinomycin (SPEC), an aminocyclitol antibiotic that targets the 30S subunit and inhibits translocation of tRNAs [69]. Despite a different mechanism of action from the other inhibitors, the biofilm stimulation response to SPEC was also insensitive to nitrate. ...
Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E . coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA , nuoE , or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon–controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a ‘pre-activated’ phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.
... Translation of mRNA nucleotide sequences to amino acids during the ribosomal synthesis of proteins is a central evolutionarily conserved cellular process that has been extensively targeted with antibiotics treating bacterial infections (10), but Plasmodium translation has not been targeted by any clinically approved antimalarials to date. Plasmodium protein synthesis is a highly desirable process to target, as translation can be blocked via many different molecular targets. ...
Plasmodium parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the Plasmodium berghei- HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypeptide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual P. berghei liver stage parasites and host cells simultaneously. After validation, we demonstrate specific, concentration-dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between known direct translation inhibitors and other compounds which we identify as indirect translation inhibitors. Using this framework, we identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in P. berghei liver stages and confirm this potential mode of action in Plasmodium falciparum asexual blood stages.
IMPORTANCE
Plasmodium parasites cause malaria in humans. New multistage active antimalarial drugs are needed, and a promising class of drugs targets the core cellular process of translation, which has many potential molecular targets. During the obligate liver stage, Plasmodium parasites grow in metabolically active hepatocytes, making it challenging to study core cellular processes common to both host cells and parasites, as the signal from the host typically overwhelms that of the parasite. Here, we present and validate a flexible assay to quantify Plasmodium liver stage translation using a technique to fluorescently label the newly synthesized proteins of both host and parasite followed by computational separation of their respective nascent proteomes in confocal image sets. We use the assay to determine whether a test set of known compounds are direct or indirect liver stage translation inhibitors and show that the assay can also predict the mode of action for novel antimalarial compounds.
... A preponderance of naturally evolved antibacterial agents and many synthetic drugs employed clinically for the treatment of human infectious diseases target the bacterial ribosome and thereby inhibit bacterial protein synthesis [1][2][3] . Among these, entire classes of chemically distinct antibiotics bind in or near the peptidyl transferase center (PTC), located within the large subunit of the bacterial ribosome, and often inhibit protein synthesis by direct competition with the binding of aminoacyl-tRNA (aa-tRNA) substrates. ...
The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.
... result in erroneous peptides, leading to aggregation products, cell death, or neurodegenerative diseases 8,9 . The essentiality of faithful tRNA selection to cellular viability is highlighted by the functions of clinically important antibiotics that specifically impair the ribosome's selectivity [10][11][12] . Such considerations have fueled extensive efforts to gain a deeper, fundamental understanding of how ribosomes achieve fidelity to thereby establish the genetic code. ...
Accurate protein synthesis is determined by the two-subunit ribosome’s capacity to selectively incorporate cognate aminoacyl-tRNA for each mRNA codon. The molecular basis of tRNA selection accuracy, and how fidelity can be affected by antibiotics, remains incompletely understood. Using molecular simulations, we find that cognate and near-cognate tRNAs delivered to the ribosome by Elongation Factor Tu (EF-Tu) can follow divergent pathways of motion into the ribosome during both initial selection and proofreading. Consequently, cognate aa-tRNAs follow pathways aligned with the catalytic GTPase and peptidyltransferase centers of the large subunit, while near-cognate aa-tRNAs follow pathways that are misaligned. These findings suggest that differences in mRNA codon-tRNA anticodon interactions within the small subunit decoding center, where codon-anticodon interactions occur, are geometrically amplified over distance, as a result of this site’s physical separation from the large ribosomal subunit catalytic centers. These insights posit that the physical size of both tRNA and ribosome are key determinants of the tRNA selection fidelity mechanism.
... Target alteration is also seen for the aminoglycosides group of antibiotics that are protein synthesis inhibitors and these antibiotics are divided into 4,6-disubstituted 2-deoxystreptamin (DOS), 4,5 disubstituted DOS and 4-monosubstituted DOS. These antibiotics have explicit effectivity after coupling with the helix 16S and 23S rRNA of the ribosomal subunit of bacteria that induces translational misinterpretation and stoppage of reactions associated with translocation [67][68][69] . Different genes are associated with developing self-resistance in different aminoglycoside antibiotics. ...
... Tetracycline is an elongation factor inhibitor that inhibits protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site [60,61]. Sub-inhibitory concentrations of translation inhibitors are known to slow down translation, but not transcription, and thus reduce transcription-translation coupling [62e64]. ...
... This suggests that partial disruption of coupled transcriptiontranslation, potentially by interfering with ribosome progression via R-loop formation, allows DssrA cells to grow in the presence of DNA damage. In support of this, we also observed that partial disruption of translation using sub-inhibitory concentrations of tetracycline, an elongation inhibitor [60,61], similarly allowed SsrAdefective cells to grow in the presence of DNA damage. ...
DNA integrity in bacteria is regulated by various factors that act on the DNA. trans-translation has previously been shown to be important for the survival of E. coli cells exposed to certain DNA-damaging agents. However, the mechanisms underlying this sensitivity are poorly understood. In this study, we explored the involvement of the trans-translation system in the maintenance of genome integrity using various DNA-damaging agents and mutant backgrounds. Relative viability assays showed that SsrA-defective cells were sensitive to DNA-damaging agents, such as nalidixic acid (NA), ultraviolet radiation (UV), and methyl methane sulfonate (MMS). The viability of SsrA-defective cells was rescued by deleting sulA, although the expression of SulA was not more pronounced in SsrA-defective cells than in wild-type cells. Live cell imaging using a Gam-GFP fluorescent reporter showed increased double-strand breaks (DSBs) in SsrA-defective cells during DNA damage. We also showed that the ribosome rescue function of SsrA was sufficient for DNA damage tolerance. DNA damage sensitivity can be alleviated by partial uncoupling of transcription and translation by using sub-lethal concentrations of ribosome inhibiting antibiotic (tetracycline) or by mutating the gene coding for RNase H (rnhA). Taken together, our results highlight the importance of trans-translation system in maintaining genome integrity and bacterial survival during DNA damage.
... Extensive efforts over the past two decades have led to antibioticribosome structures for every major class of clinically used ribosometargeting antibiotic, including aminoglycosides, tetracyclines, lincosamides, macrolides, oxazolidinones, pleuromutilins, streptomycins and spectinomycins, thereby providing fundamental insight into their binding sites and mechanisms of action [1][2][3][4][5][6] . Generally, most of these antibiotic-ribosome structures are reported at resolutions ranging from 2.5 to 3.5 Å, with a few recent studies obtaining resolutions below 2.5 Å (refs. ...
The ribosome is a major target for clinically used antibiotics, but multidrug resistant pathogenic bacteria are making our current arsenal of antimicrobials obsolete. Here we present cryo-electron-microscopy structures of 17 distinct compounds from six different antibiotic classes bound to the bacterial ribosome at resolutions ranging from 1.6 to 2.2 Å. The improved resolution enables a precise description of antibiotic–ribosome interactions, encompassing solvent networks that mediate multiple additional interactions between the drugs and their target. Our results reveal a high structural conservation in the binding mode between antibiotics with the same scaffold, including ordered water molecules. Water molecules are visualized within the antibiotic binding sites that are preordered, become ordered in the presence of the drug and that are physically displaced on drug binding. Insight into RNA–ligand interactions will facilitate development of new antimicrobial agents, as well as other RNA-targeting therapies.
... Despite this, albocycline may represent a solution for the treatment of infections caused by S. aureus species. A structural motif in the macrolide family of antibiotics, the 14-membered macrolactone of albocycline indicates that it targets the bacterial ribosome and thereby inhibits translation [84]. ...
This narrative review paper provides an up-to-date overview of the potential of novel synthetic and semisynthetic compounds as antibacterials that target virulence traits in resistant strains. The review focused on research conducted in the last five years and investigated a range of compounds including azoles, indoles, thiophenes, glycopeptides, pleuromutilin derivatives, lactone derivatives, and chalcones. The emergence and spread of antibiotic-resistant bacterial strains is a growing public health concern, and new approaches are urgently needed to combat this threat. One promising approach is to target virulence factors, which are essential for bacterial survival and path-ogenesis, but not for bacterial growth. By targeting virulence factors, it may be possible to reduce the severity of bacterial infections without promoting the development of resistance. We discuss the mechanisms of action of the various compounds investigated and their potential as antibacterials. The review highlights the potential of targeting virulence factors as a promising strategy to combat antibiotic resistance and suggests that further research is needed to identify new compounds and optimize their efficacy. The findings of this review suggest that novel synthetic and semisynthetic compounds that target virulence factors have great potential as antibacterials in the fight against antibiotic resistance.
... Other chemical tools led us to investigate the nature of pep-tRNA drop-off. Macrolides are antibiotics that bind in the nascent peptide exit tunnel of the ribosome and inhibit protein synthesis 58 . The macrolide erythromycin stimulates the dissociation of pep-tRNA and increases the temperature sensitivity of pth ts cells 41,59 . ...
In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
