Oxazolidinones Mechanism of Action: Inhibition of the First Peptide Bond Formation

Patel, Utpal; Yan, Yong Ping; Hobbs, Frank W.; Kaczmarczyk, Janet; Slee, Andrew M.; Pompliano, David L.; Kurilla, Michael G.; Bobkova, Ekaterina V.

doi:10.1074/jbc.m102966200

Cited by 51 publications

(28 citation statements)

References 27 publications

(25 reference statements)

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“…However, the ability of oxazolidinones to inhibit initiation complex formation has been observed only at high ratios of oxazolidinones to ribosomes. Moreover, attempts to demonstrate that oxazolidinones inhibit peptide bond formation have not produced clear results, except for the inhibition of the first peptide bond as a consequence of the interference of linezolid with the binding of initiator fMet-tRNA(i)(Met) (35). Alternative mechanisms of oxazolidinone action have been proposed when experiments showed that oxazolidinones had a significant in vivo effect on frameshifting and nonsense suppression at concentrations below the MIC (48) and were also able to inhibit 50S ribosomal subunit assembly (8).…”

Section: Discussionmentioning

confidence: 99%

Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

Skripkin

McConnell

DeVito

et al. 2008

Antimicrob Agents Chemother

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New and improved antibiotics are urgently needed to combat the ever-increasing number of multidrugresistant bacteria. In this study, we characterized several members of a new oxazolidinone family, R-01. This antibiotic family is distinguished by having in vitro and in vivo activity against hospital-acquired, as well as community-acquired, pathogens. We compared the 50S ribosome binding affinity of this family to that of the only marketed oxazolidinone antibiotic, linezolid, using chloramphenicol and puromycin competition binding assays. The competition assays demonstrated that several members of the R-01 family displace, more effectively than linezolid, compounds known to bind to the ribosomal A site. We also monitored binding by assessing whether R-01 compounds protect U2585 (Escherichia coli numbering), a nucleotide that influences peptide bond formation and peptide release, from chemical modification by carbodiimide. The R-01 oxazolidinones were able to inhibit translation of ribosomes isolated from linezolid-resistant Staphylococcus aureus at submicromolar concentrations. This improved binding corresponds to greater antibacterial activity against linezolid-resistant enterococci. Consistent with their ribosomal A-site targeting and greater potency, the R-01 compounds promote nonsense suppression and frameshifting to a greater extent than linezolid. Importantly, the gain in potency does not impact prokaryotic specificity as, like linezolid, the members of the R-01 family show translation 50% inhibitory concentrations that are at least 100-fold higher for eukaryotic than for prokaryotic ribosomes. This new family of oxazolidinones distinguishes itself from linezolid by having greater intrinsic activity against linezolid-resistant isolates and may therefore offer clinicians an alternative to overcome linezolid resistance. A member of the R-01 family of compounds is currently undergoing clinical trials.

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Section: Discussionmentioning

confidence: 99%

Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

Skripkin

McConnell

DeVito

et al. 2008

Antimicrob Agents Chemother

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“…The fragment P-site substrate CCA-Phe-X-Biotin was synthesized as described in Ref. 1 Preparation of Oxazolidinone-resistant 23 S RNA Mutant Ribosomes-Oxazolidinone-resistant mutant strains carrying a single mutated copy of ribosomal 23 S RNA were selected and characterized as reported in Refs. 2 and 33 using plasmids with site-directed mutations (10) and a recently described single plasmid-encoded rRNA operon system in Escherichia coli (11).…”

Section: Reagents and Materials-puromycin Chloramphenicol And Trna Imentioning

confidence: 99%

“…Oxazolidinones, the only novel class of antibiotics identified in the last two decades, are the focus of intensive discovery efforts (1)(2)(3)(4)(5)(6)(7)(8)(9). Linezolid, an oxazolidinone, is approved for treatment of infections caused by Gram-positive bacteria that are resistant to other antibiotics.…”

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confidence: 99%

Catalytic Properties of Mutant 23 S Ribosomes Resistant to Oxazolidinones

Bobkova

Yan²,

Jordan

et al. 2003

Journal of Biological Chemistry

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Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of K m for initiator tRNA and a 100-fold decrease in V max in puromycin reaction. Comparison of the K i values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrateindependent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P-and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.Oxazolidinones, the only novel class of antibiotics identified in the last two decades, are the focus of intensive discovery efforts (1-9). Linezolid, an oxazolidinone, is approved for treatment of infections caused by Gram-positive bacteria that are resistant to other antibiotics. Emerging resistance to all known drugs, including the "last resort" vancomycin family, poses a serious threat to the public health worldwide. Understanding the mechanism of action of oxazolidinones at the molecular level, therefore, has a great importance for the development of the next generation of these novel antibiotics and, ultimately, for the outcome of the ongoing battle against drug-resistant pathogens.Oxazolidinones impose their action at the initiation stage of translation (4, 5), apparently via inhibition of preinitiation complex formation (9). Our recent finding that oxazolidinones interfere with binding of initiator tRNA to the ribosomal P-site (1), thus inhibiting formation of the first peptide bond, prompted a search for similarities between oxazolidinones and known inhibitors of peptidyltransferase. To address this and other mechanistic questions, we have studied catalytic properties of oxazolidinone-resistant ribosomes and compared the mechanism of oxazolidinone inhibition with the action of known peptidyltransferase inhibitors, such as chloramphenicol and sparsomycin. To further define the oxazolidinone binding site, we have mapped resistant mutations onto the three-dimensional structure of the ribosomal 50 S subunit to reveal a...

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“…Oxazolidinones inhibit bacterial protein synthesis via competitive binding to the 23S rRNA in the catalytic site of the bacterial 50S ribosomes (3,4). Linezolid (LZD), the first oxazolidinone that entered clinical use, exhibits bacteriostatic activity against M. tuberculosis with an MIC of less than 1 g/ml (5) and has been used against MDR/XDR TB cases (4,6,7). However, long-term administration of LZD may cause severe side effects such as anemia, thrombocytopenia, and peripheral neuropathy (8).…”

mentioning

confidence: 99%

In Vitro and In Vivo Activities of Three Oxazolidinones against Nonreplicating Mycobacterium tuberculosis

Zhang

Sala

Dhar

et al. 2014

Antimicrob Agents Chemother

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cOxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starved Mycobacterium tuberculosis strain 18b. The in vitro drug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection with M. tuberculosis 18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis. Despite the availability of effective chemotherapy, tuberculosis (TB) is still a major global health problem, and Ͼ8.6 million cases of active disease and 1.4 million deaths were reported in 2012 (1). Moreover, approximately one-third of the world's population is latently infected with Mycobacterium tuberculosis and at risk of reactivation (1). The slow growth and dormancy of M. tuberculosis contribute to the chronic nature of the infection, and effective treatment of active disease requires lengthy multidrug regimens. This situation is now worsened by the increasing emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even totally drug-resistant M. tuberculosis strains (1), thus underscoring the urgent need for new molecules with potent activity against latent M. tuberculosis and no cross-resistance with existing therapeutic agents.Oxazolidinones are one of the only three new classes of antibiotics that have been approved by the Food and Drug Administration (FDA) in the last 35 years (2). Oxazolidinones inhibit bacterial protein synthesis via competitive binding to the 23S rRNA in the catalytic site of the bacterial 50S ribosomes (3, 4). Linezolid (LZD), the first oxazolidinone that entered clinical use, exhibits bacteriostatic activity against M. tuberculosis with an MIC of less than 1 g/ml (5) and has been used against MDR/XDR TB cases (4, 6, 7). However, long-term administration of LZD may cause severe side effects such as anemia, thrombocytopenia, and peripheral neuropathy (8). PNU-100480 (sutezolid; PNU) is an oxazolidinone analogue that has been reported to have an improved safety profile compared to LZD (2). PNU has an MIC similar to that of LZD for M. tuberculosis in vitro but has more potent bactericidal activity in ex vivo whole-blood cultures and in a murine model of TB (9-11). In mice, addition of PNU not only enhanced the bactericidal activity during the initial ph...

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Oxazolidinones Mechanism of Action: Inhibition of the First Peptide Bond Formation

Cited by 51 publications

References 27 publications

Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

Catalytic Properties of Mutant 23 S Ribosomes Resistant to Oxazolidinones

In Vitro and In Vivo Activities of Three Oxazolidinones against Nonreplicating Mycobacterium tuberculosis

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