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Discovery of small-molecule inhibitors targeting the ribosomal peptidyl transferase center (PTC) of M. tuberculosis.

ABSTRACT: M. tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis, which kills more than 1.5 million people worldwide every year. Strains resistant to available antibiotics pose a significant healthcare problem. The enormous complexity of the ribosome poses a barrier for drug discovery. We have overcome this in a tractable way by using an RNA segment that represents the peptidyl transferase center as a target. By using a novel combination of NMR transverse relaxation times (T 2) and computational chemistry approaches, we have obtained improved inhibitors of the Mtb ribosomal PTC. Two phenylthiazole derivatives were predicted by machine learning models as effective inhibitors, and this was confirmed by their IC50 values, which were significantly improved over standard antibiotic drugs.

SUBMITTER: Tam B

PROVIDER: S-EPMC6849635 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Discovery of small-molecule inhibitors targeting the ribosomal peptidyl transferase center (PTC) of M. tuberculosis.

Tam Benjamin B Sherf Dror D Cohen Shira S Eisdorfer Sarah Adi SA Perez Moshe M Soffer Adam A Vilenchik Dan D Akabayov Sabine Ruth SR Wagner Gerhard G Akabayov Barak B

Chemical science 20190806 38

M. tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis, which kills more than 1.5 million people worldwide every year. Strains resistant to available antibiotics pose a significant healthcare problem. The enormous complexity of the ribosome poses a barrier for drug discovery. We have overcome this in a tractable way by using an RNA segment that represents the peptidyl transferase center as a target. By using a novel combination of NMR transverse relaxation times (T</i ...[more]

PMID: 31803448

Similar Datasets

Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

Project description:We report the context-specific activity of two peptidyl transferase targeting antibiotics, chloramphenicol and linezolid. By generating ribosome profiling data in the presence or absence of either chloramphenicol or linezolid we mapped the relative change of ribosome density induced by these antibiotics. We find that both inhibitors preferentially arrest ribosomes that carry either an alanine, serine, or threonine in the penultimate (-1) position of the nascent peptide chain. Additionally ribosomes that carry a glycine in either the P site (0) or A-site (+1) counteract the inhibitory activity of both inhibitors. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.

2016-09-08 | GSE86536 | GEO

Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center.

Project description:The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.

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Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center.

Project description:Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.

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The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning.

Project description:The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.

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A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center.

Project description:The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg(2+)-muc's). Common features of Mg(2+)-muc's include two paired Mg(2+) ions that are chelated by a common bridging phosphate group in the form Mg((a))(2+)-(O1P-P-O2P)-Mg((b))(2+). This bridging phosphate is part of a 10-membered chelation ring in the form Mg((a))(2+)-(OP-P-O5'-C5'-C4'-C3'-O3'-P-OP)-Mg((a))(2+). The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg(2+) ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg(2+)-muc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg(2+)-muc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg(2+)-muc's in other rRNAs including the bacterial 16S rRNA, and the P4-P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg(2+)-muc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.

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The DEAD-box protein Dbp6 is an ATPase and RNA annealase interacting with the peptidyl transferase center (PTC) of the ribosome.

Project description:Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA-RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle. We demonstrate that in vitro, Dbp6 shows ATPase as well as annealing and clamping activities negatively regulated by ATP. Mutations in Dbp6 core motifs involved in ATP binding and ATP hydrolysis are lethal and impair Dbp6 ATPase activity but increase its RNA binding and RNA annealing activities. These data suggest that correct regulation of these activities is important for Dbp6 function in vivo. Using in vivo cross-linking (CRAC) experiments, we show that Dbp6 interacts with 25S rRNA sequences located in the 5' domain I and in the peptidyl transferase center (PTC), and also crosslinks to snoRNAs hybridizing to the immature PTC. We propose that the ATPase and RNA clamping/annealing activities of Dbp6 modulate interactions of snoRNAs with the immature PTC and/or contribute directly to the folding of this region.

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The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center.

Project description:Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.

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Single-molecule study of ribosome hierarchic dynamics at the peptidyl transferase center.

Project description:During protein biosynthesis the ribosome moves along mRNA in steps of precisely three nucleotides. The mechanism for this ribosome motion remains elusive. Using a classification algorithm to sort single-molecule fluorescence resonance energy transfer data into subpopulations, we found that the ribosome dynamics detected at the peptidyl transferase center are highly inhomogeneous. The pretranslocation complex has at least four subpopulations that sample two hybrid states, whereas the posttranslocation complex is mainly static. We observed transitions among the ribosome subpopulations under various conditions, including 1), in the presence of EF-G; 2), spontaneously; 3), in different buffers, and 4), bound to antibiotics. Therefore, these subpopulations represent biologically active ribosomes. One key observation indicates that the Hy2 hybrid state only exists in a fluctuating ribosome subpopulation, which prompts us to propose that ribosome dynamics are hierarchically arranged. This proposal may have important implications for the regulation of cellular translation rates.

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Peptidyl Transferase Center and the Emergence of the Translation System.

Project description:In this work, the three-dimensional (3D) structure of the ancestral Peptidyl Transferase Center (PTC) built by concatamers of ancestral sequences of tRNAs was reconstructed, and its possible interactions with tRNAs molecules were analyzed. The 3D structure of the ancestral PTC was also compared with the current PTC of T. thermophilus. Docking experiments between the ancestral PTC and tRNAs suggest that in the origin of the translation system, the PTC functioned as an adhesion center for tRNA molecules. The approximation of tRNAs charged with amino acids to the PTC permitted peptide synthesis without the need of a genetic code.

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The 2'-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

Project description:The ribosome accelerates the rate of peptidyl transfer by >10(6)-fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2'-OH of the peptidyl-tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2'-OH of the peptidyl-tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100-fold slower with the dA76-substituted peptidyl-tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl-tRNA lacks the 2'-OH group. Additionally, the 2'-OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and support a model where interactions between active site residues and the 2'-OH of A76 of the peptidyl-tRNA are pivotal in orienting substrates in this active site for optimal catalysis.

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