Dean L. Shinabarger

In Vitro Activity of TR-700, the Antibacterial Moiety of the Prodrug TR-701, against Linezolid-Resistant Strains

ABSTRACT TR-701 is the orally active prodrug of TR-700, a novel oxazolidinone that demonstrates four- to eightfold-greater activity than linezolid (LZD) against Staphylococcus and Enterococcus spp. In this study evaluating the in vitro sensitivity of LZD-resistant isolates, TR-700 demonstrated 8- to 16-fold-greater potency than LZD against all strains tested, including methicillin-resistant Staphylococcus aureus (MRSA), strains of MRSA carrying the mobile cfr methyltransferase gene, and vancomycin-resistant enterococci. The MIC90 for TR-700 against LZD-resistant S. aureus was 2 μg/ml, demonstrating the utility of TR-700 against LZD-resistant strains. A model of TR-700 binding to 23S rRNA suggests that the increased potency of TR-700 is due to additional target site interactions and that TR-700 binding is less reliant on target residues associated with resistance to LZD.

Oxazolidinone Antibiotics Target the P Site on Escherichia coli Ribosomes

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 Halobacteriumhalobium, Staphylococcusaureus, and Escherichiacoli 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.

In Vitro Activity of TR-700, the Active Ingredient of the Antibacterial Prodrug TR-701, a Novel Oxazolidinone Antibacterial Agent

ABSTRACT TR-701 is the prodrug of the microbiologically active molecule TR-700, a novel orally and intravenously administered oxazolidinone antibacterial agent. The in vitro activity of TR-700 was evaluated against 1,063 bacterial clinical isolates including staphylococci, enterococci, streptococci, Moraxella catarrhalis, Haemophilus influenzae, and a variety of anaerobic bacterial species. The test strains were recent (2005 to 2008) clinical isolates from diverse U.S. (80%) and non-U.S. (20%) sites. MIC assays were conducted using reference broth microdilution and agar dilution methods with the principal comparators linezolid and vancomycin. TR-700 was four- to eightfold more potent than linezolid against staphylococci and generally fourfold more potent against enterococci and streptococci. TR-700 was less active against M. catarrhalis and H. influenzae but was twofold more active than linezolid. Against anaerobic species, the activity of TR-700 was equivalent to or up to fourfold higher than that of linezolid. These results indicate that TR-700 is a promising new oxazolidinone antibacterial agent with greater in vitro potency than linezolid against clinically important gram-positive bacteria.

Biosynthesis of the Glycolipid Anchor in Lipoteichoic Acid of Staphylococcus aureus RN4220: Role of YpfP, the Diglucosyldiacylglycerol Synthase

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419-430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either L- or D-alanine. Moreover, viability of the mutant at 37 degrees C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

1H Nuclear Magnetic Resonance Study of Oxazolidinone Binding to Bacterial Ribosomes

ABSTRACT The oxazolidinones are a novel class of antibiotics that inhibit initiation of protein synthesis in bacteria. In order to investigate their novel mechanism of action, the interactions of several oxazolidinones with bacterial 70S ribosomes, 50S subunits, and 30S subunits have been characterized by 1H nuclear magnetic resonance (NMR) line-broadening analyses and transferred nuclear Overhauser enhancement (TRNOE) experiments. PNU-177553 and PNU-100592 (eperezolid) and their corresponding enantiomers, PNU-184414 and PNU-107112, were studied. The dissociation constants were determined to be 94 ± 44 μM and 195 ± 40 μM for PNU-177553 and eperezolid, respectively. There was a ∼4-fold decrease in affinity for their corresponding enantiomers. The NMR-derived dissociation constants are consistent with their antibacterial activity. PNU-177553 and eperezolid were found to bind only to the 50S subunit, with similar affinity as to the 70S ribosome, and to have no affinity for the 30S subunit. Specific binding of PNU-177553 was further confirmed in TRNOE experiments in which positive NOEs observed for the small molecule alone were changed to negative NOEs in the presence of bacterial 70S ribosomes. The observed NOEs indicated that PNU-177553 did not adopt a significantly different conformation when bound to the 70S ribosome, compared to the extended conformation that exists when free in solution. Since this is likeliest the case for each of the four compounds included in this study, the A ring C5 side chain may be positioned in the proper orientation for antibacterial activity in PNU-177553 and eperezolid but not in their inactive enantiomers.

Comparative in vitro activity profiles of novel bis-indole antibacterials against gram-positive and gram-negative clinical isolates.

ABSTRACT Antimicrobial susceptibilities of 233 Gram-positive and 180 Gram-negative strains to two novel bis-indoles were evaluated. Both compounds were potent inhibitors of Gram-positive bacteria, with MIC90 values of 0.004 to 0.5 μg/ml. One bis-indole, MBX 1162, exhibited potent activity against all Gram-negative strains, with MIC90 values of 0.12 to 4 μg/ml, even against high-level-resistant pathogens, and compared favorably to all comparator antibiotics. The bis-indole compounds show promise for the treatment of multidrug-resistant clinical pathogens.

Characterization of a High-Throughput Screening Assay for Inhibitors of Elongation Factor P and Ribosomal Peptidyl Transferase Activity

Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent Ka of 0.75 μM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent Kms of 19 μM and 0.5 μM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.

Comparative in vitro activity of oritavancin and other agents against methicillin-susceptible and methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a threat to the public health due to their prevalence and associated mortality and morbidity. Several agents have been recently approved to treat MRSA skin infections including lipoglycopeptides (dalbavancin, oritavancin, and telavancin), ceftaroline, and tedizolid. This study compared the MIC, minimum bactericidal concentration (MBC), and time-kill of these agents alongside daptomycin, linezolid, and vancomycin against MRSA (n=15); meropenem, cefazolin, and nafcillin were also included against methicillin-susceptible S. aureus (MSSA [n=12]). MIC and MBC testing was conducted in accordance with Clinical and Laboratory Standards Institute guidelines, and time-kills were evaluated at multiples of the MIC and the free-drug maximum plasma concentration (fCmax) at both standard and high inoculum densities for a subset of MRSA (n=2) and MSSA (n=2). MRSA and MSSA were highly susceptible to all agents, with the lipoglycopeptides having the most potent activity by MIC50/90. All agents excluding tedizolid and linezolid were bactericidal by MBC for MRSA and MSSA, though dalbavancin and telavancin exhibited strain-specific bactericidal activity for MRSA. All agents excluding tedizolid and linezolid were bactericidal by time-kill at their respective fCmax against MRSA and MSSA at standard inoculum density, though oritavancin exhibited the most rapid bactericidal activity. Oritavancin and daptomycin at their respective fCmax maintained similar kill curves at high inoculum density. In contrast, the killing observed with other agents was typically reduced or slowed at high inoculum density. These data demonstrate the rapid bactericidal activity of oritavancin and daptomycin against S. aureus relative to other MRSA agents regardless of bacterial burden.

Comparative in vitro activity of oritavancin and other agents against vancomycin-susceptible and -resistant enterococci

Sir, Antibiotic research has focused on discovering agents with activity against MDR pathogens (e.g. ESKAPE pathogens), including VRE, which along with Staphylococcus aureus are commonly isolated from healthcare-associated infections. Several new agents have been approved for the treatment of skin and skin structure infections caused by MRSA and enterococci, including oxazolidinones (linezolid and tedizolid), lipoglycopeptides (oritavancin, dalbavancin and telavancin), a cyclic lipopeptide (daptomycin), a glycylcycline (tigecycline) and an anti-MRSA cephalosporin (ceftaroline). Lipoglycopeptides, though active against vancomycin-susceptible enterococci (VSE), have variable activity against VRE, with oritavancin being the sole agent maintaining potent activity against VanA-type VRE. We report here a direct comparison of the in vitro activity of lipoglycopeptides and other skin agents against vancomycinsusceptible and -resistant Enterococcus faecalis (VSEfa and VREfa) and Enterococcus faecium (VSEfm and VREfm). Comparative evaluations included MIC and MBC determinations and time–kill kinetics. Since variation in inoculum density has been shown to impact the activity of several of these agents against S. aureus, time–kill kinetics were assessed at both standard and high inoculum densities. The evaluated isolates consisted of 74 random non-duplicate clinical isolates of VSEfa, VREfa, VSEfm and VREfm from the Micromyx repository (Kalamazoo, MI, USA) and The Medicines Company (Ville Saint Laurent, Quebec, Canada). VanA-phenotype (vancomycin and teicoplanin resistant) and VanB-phenotype (vancomycin resistant and teicoplanin susceptible) VRE were selected based on prior glycopeptide susceptibility test history. Agents were handled per CLSI (formerly NCCLS) guidelines and had results within CLSI quality control ranges during testing. Evaluations of lipoglycopeptides incorporated polysorbate 80 at a final concentration of 0.002% (v/v). MIC and MBC values were determined in accordance with standard CLSI methods. The time–kill kinetics of select isolates (one per phenotype evaluated) at standard inoculum ( 5 10 cfu/ mL) and high inoculum ( 5 10 cfu/mL) were determined as described by Arhin et al. using a method derived from the CLSI for agents at their fCmax (calculated from the respective prescribing information as 16 mg/L for oritavancin, dalbavancin, linezolid, vancomycin and ceftaroline, 8 mg/L for telavancin, 4 mg/L for daptomycin and 1 mg/L for tedizolid). The activity of the tested agents against enterococci is summarized by species and phenotype in Table 1. Among E. faecalis, VSEfa were susceptible to all agents with the lipoglycopeptides having the most potent activity by MIC90. Ceftaroline and oritavancin were the only consistently bactericidal agents against VSEfa based on the proportion of isolates with MBC:MIC ratios of 4. Based on MIC90, oritavancin, daptomycin, linezolid, tedizolid and ceftaroline maintained potent activity against VanA VREfa. Ceftaroline maintained bactericidal activity against VanA VREfa, while the other agents typically had MBC:MIC ratios>4. Against VSEfm and VREfm (VanA and VanB phenotypes), oritavancin was the most potent agent evaluated based on MIC90. All agents, excluding ceftaroline, which was largely inactive against E. faecium, had potent activity against VSEfm by MIC90. Oritavancin, daptomycin, linezolid and tedizolid maintained potent activity against VREfm. Oritavancin was 16-fold more potent by MIC90 than the comparator lipoglycopeptides against VREfm. Against VSEfm, oritavancin and daptomycin were the only consistently bactericidal agents based on the proportion of isolates with MBC:MIC ratios of 4. Daptomycin maintained bactericidal activity by MBC:MIC ratio against VanA and VanB VREfm. Consistent trends in bactericidal activity were apparent for each agent by time–kill at fCmax across the evaluated E. faecalis (one isolate each of VSEfa and VanA VREfa) and E. faecium (one isolate each of VSEfm, VanA VREfm and VanB VREfm) isolates. At the standard inoculum density, oritavancin and daptomycin were rapidly bactericidal with 3 log killing typically achieved within 0.25 and 4 h, respectively, with singular exceptions (4 h for oritavancin and 1 h for daptomycin against the VanA VREfa isolate). Telavancin was typically bactericidal, with 3 log killing observed at 24 h at the standard inoculum density with the exception of the VanB VREfm isolate, as was ceftaroline for E. faecalis but not E. faecium. Vancomycin, dalbavancin, tedizolid and linezolid did not achieve 3 log killing at the standard inoculum density for any of the evaluated E. faecalis or E. faecium isolates. Only oritavancin maintained bactericidal activity at the high inoculum density, with similar kill kinetics to those observed at the standard inoculum density. Few available agents exist for treating VRE infections despite their prevalence and associated mortality and morbidity. Among agents with activity against enterococci, oritavancin, daptomycin, linezolid and tedizolid maintained potent activity against VanA/ VanB VRE isolates. Both daptomycin and oritavancin exhibited rapid bactericidal activity at fCmax against VSE and VRE at standard inoculum densities, but only oritavancin maintained rapid

Evaluation of the bactericidal activity of Telavancin against Staphylococcus aureus using revised testing guidelines

The in vitro broth microdilution testing method for telavancin, a lipoglycopeptide active against S. aureus, was revised in 2014 to include polysorbate-80 in the test media. This study evaluates the bactericidal activity of telavancin against S. aureus in media containing polysorbate-80 by in vitro time-kill analysis alongside relevant comparators.