Simultaneous determination of a novel oxazolidinone anti-tuberculosis OTB-658 and its metabolites in monkey blood by LC-MS/MS.

OTB-658, a novel oxazolidinone anti-tuberculosis agent, has potent antibacterial activity against Mycobacterium tuberculosis, especially multi-drug resistant tuberculosis (MDR-TB) in vitro and in vivo. In this study, after metabolite identification of parent drug OTB-658, a specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established and validated to quantify OTB-658 and its metabolites OTB-665 and OTB-698 in monkey blood. HHY-1442, an analogue compound of OTB-658, was used as the internal standard. Blood samples were prepared by direct protein precipitation. Separation was performed on a Zorbax SB C18 column (50 mm × 2.1 mm, 3.5 µm) with a gradient mobile phase of methanol/water at a flow rate of 0.3 mL/min. The detection was conducted by a positive electrospray ionization in multiple-reaction monitoring mode on a triple quadrupole MS. The monitored transitions were m/z 382.2 â†’ 221.1 for OTB-658, m/z 398.2 â†’ 308.1 for OTB-665, m/z 414.1 â†’ 372.3 for OTB-698 and m/z 418.2 â†’ 311.3 for HHY-1442, respectively. Good linearity was observed over the range of 10-2000 ng/mL for OTB-658 and OTB-665, and 5-1000 ng/mL for OTB-698. All the intra-day and inter-day precision for the three analytes was below 8.4%, and the accuracy ranged from 96.0% to 106.0%. All analytes were stable during storage, preparation, and analytical procedures. The validated method was successfully applied to pharmacokinetic and bioavailability studies of OTB-658 in cynomolgus monkeys and the absolute bioavailability of OTB-658 was 25.0% at an oral dose of 10 mg/kg.

Pharmacokinetic Drug Interactions of an Orally Available TRH Analog (Rovatirelin) With a CYP3A4/5 and P-Glycoprotein Inhibitor (Itraconazole).

The effects of itraconazole on the pharmacokinetics of rovatirelin were investigated in an open-label, single-sequence drug-drug interaction study in 16 healthy subjects. Subjects were administered a single oral dose of rovatirelin (1.6 mg) on day 1 and day 15. From day 8 through 16, subjects received daily oral doses of itraconazole (200 mg/day). Concentrations of rovatirelin and (thiazolylalanyl)methylpyrrolidine (TAMP), the major metabolite of rovatirelin formed by cytochrome P450 (CYP) 3A4/5, were determined in plasma and urine. Pharmacokinetic parameters were used to evaluate the drug-drug interaction potential of rovatirelin as a victim. With coadministration, maximum concentration (Cmax ) and area under the concentration-time curve extrapolated to infinity (AUCinf ) of rovatirelin increased 3.05-fold and 2.82-fold, respectively, and the 90% confidence intervals of the ratios for Cmax (2.64-3.52) and AUCinf (2.47-3.23) did not fall within the 0.8-1.25 boundaries. Urinary excretion of rovatirelin increased at almost the same ratio as the AUCinf ratio with coadministration; however, renal clearance did not change. Cmax , AUCinf , and urinary excretion of TAMP were decreased by coadministration. Itraconazole has the potential to inhibit drug transport via intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP); therefore, substrate assessments of rovatirelin for the 2 transporters were evaluated using Caco-2 cell monolayers. In vitro studies showed that rovatirelin is a substrate for P-gp but not for BCRP. The current study shows that itraconazole's effect on rovatirelin pharmacokinetics is mediated through inhibition of CYP3A4/5 and intestinal P-gp.

Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.

Synthesis and antibacterial activity evaluation of novel biaryloxazolidinone analogues containing a hydrazone moiety as promising antibacterial agents.

A series of linezolid analogues containing a hydrazone moiety were designed, synthesized and evaluated for their antibacterial activity. Most compounds exhibited more potent antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens as compared with linezolid and radezolid. Compounds 9a, 9c, 9f, 9g, 10m and 10t were more potent against tested clinical isolates of MRSA, MSSA, VRE and LREF as compared to linezolid. Compound 9a exhibited comparable activity with linezolid against human MAO-A for safety evaluation and showed moderate metabolism in human liver microsome. The most promising compound 9a showed remarkable antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens with MIC value of 0.0675 mg/mL, respectively, which was 15- to 30-fold more potent than linezolid.

Evaluation of Organic Anion Transporter 1A2-knock-in Mice as a Model of Human Blood-brain Barrier.

The present study aimed to establish a humanized mouse model with which to explore OATP1A2-mediated transcellular transport of drug substrates across the blood-brain barrier (BBB) and to evaluate the usefulness of the humanized mice in preclinical studies. Sulpiride, amisulpride, sultopride, and triptans were used as probes to discriminate OATP1A2 and Oatp1a4. We generated a mouse line humanized for OATP1A2 by introducing the coding region downstream of the Oatp1a4 promoter using the CRISPR/Cas9 technique. In the mice generated, OATP1A2 mRNA in the brain was increased corresponding to disappearance of Oatp1a4. OATP1A2 was localized on both the luminal and abluminal sides of the BBB. Unfortunately, study in vivo employing sulpiride, sumatriptan, and zolmitriptan as probes did not indicate any difference in their brain-to-plasma ratio between the control and humanized mice. Quantitative targeted absolute proteomic analysis of the BBB fraction from the humanized mice revealed that almost all analyzed transporters and membrane proteins were expressed at similar levels to those in control mice. The quantitative levels of OATP1A2 differed depending on the peptide quantified, which suggests that incomplete translation or posttranslational modification may occur. The blood-to-brain transport of zolmitriptan, determined by brain perfusion in situ, was 1.6-fold higher in the humanized mice than in the controls, whereas that of sulpiride was not significantly changed. To our knowledge, we established a mouse line humanized for a BBB uptake transporter for the first time. Unfortunately, because of limited impact, there is still room for improvement of the model system.

Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A.

Protein synthesis plays an essential role in cell proliferation, differentiation, and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, (-)-agelastatin A (AglA), possesses potent antitumor activity. Its underlying mechanism of action, however, has remained unknown. Using a systematic top-down approach, we show that AglA selectively inhibits protein synthesis. Using a high-throughput chemical footprinting method, we mapped the AglA-binding site to the ribosomal A site. A 3.5 Å crystal structure of the 80S eukaryotic ribosome from S. cerevisiae in complex with AglA was obtained, revealing multiple conformational changes of the nucleotide bases in the ribosome accompanying the binding of AglA. Together, these results have unraveled the mechanism of inhibition of eukaryotic translation by AglA at atomic level, paving the way for future structural modifications to develop AglA analogs into novel anticancer agents.

Effect of rovatirelin, a novel thyrotropin-releasing hormone analog, on the central noradrenergic system.

Rovatirelin ([1-[-[(4S,5S)-(5-methyl-2-oxo oxazolidin-4-yl) carbonyl]-3-(thiazol-4-yl)-l-alanyl]-(2R)-2-methylpyrrolidine) is a novel synthetic agent that mimics the actions of thyrotropin-releasing hormone (TRH). The aim of this study was to investigate the electrophysiological and pharmacological effects of rovatirelin on the central noradrenergic system and to compare the results with those of another TRH mimetic agent, taltirelin, which is approved for the treatment of spinocerebellar degeneration (SCD) in Japan. Rovatirelin binds to the human TRH receptor with higher affinity (Ki=702nM) than taltirelin (Ki=3877nM). Rovatirelin increased the spontaneous firing of action potentials in the acutely isolated noradrenergic neurons of rat locus coeruleus (LC). The facilitatory action of rovatirelin on the firing rate in the LC neurons was inhibited by the TRH receptor antagonist, chlordiazepoxide. Reduction of the extracellular pH increased the spontaneous firing of LC neurons and rovatirelin failed to increase the firing frequency further, indicating an involvement of acid-sensitive K+ channels in the rovatirelin action. In in vivo studies, oral administration of rovatirelin increased both c-Fos expression in the LC and extracellular levels of noradrenaline (NA) in the medial prefrontal cortex (mPFC) of rats. Furthermore, rovatirelin increased locomotor activity. The increase in NA level and locomotor activity by rovatirelin was more potent and longer acting than those by taltirelin. These results indicate that rovatirelin exerts a central nervous system (CNS)-mediated action through the central noradrenergic system, which is more potent than taltirelin. Thus, rovatirelin may have an orally effective therapeutic potential in patients with SCD.

Differential responses of the antioxidant system of ametryn and clomazone tolerant bacteria.

The herbicides ametryn and clomazone are widely used in sugarcane cultivation, and following microbial degradation are considered as soil and water contaminants. The exposure of microorganisms to pesticides can result in oxidative damage due to an increase in the production of reactive oxygen species (ROS). This study investigated the response of the antioxidant systems of two bacterial strains tolerant to the herbicides ametryn and clomazone. Bacteria were isolated from soil with a long history of ametryn and clomazone application. Comparative analyses based on 16S rRNA gene sequences revealed that strain CC07 is phylogenetically related to Pseudomonas aeruginosa and strain 4C07 to P. fulva. The two bacterial strains were grown for 14 h in the presence of separate and combined herbicides. Lipid peroxidation, reduced glutathione content (GSH) and antioxidant enzymes activities were evaluated. The overall results indicated that strain 4C07 formed an efficient mechanism to maintain the cellular redox balance by producing reactive oxygen species (ROS) and subsequently scavenging ROS in the presence of the herbicides. The growth of bacterium strain 4C07 was inhibited in the presence of clomazone alone, or in combination with ametryn, but increased glutathione reductase (GR) and glutathione S-transferase (GST) activities, and a higher GSH concentration were detected. Meanwhile, reduced superoxide dismutase (SOD), catalase (CAT) and GST activities and a lower concentration of GSH were detected in the bacterium strain CC07, which was able to achieve better growth in the presence of the herbicides. The results suggest that the two bacterial strains tolerate the ametryn and clomazone herbicides with distinctly different responses of the antioxidant systems.

An immunochromatographic assay for rapid and direct detection of 3-amino-5-morpholino-2-oxazolidone (AMOZ) in meat and feed samples.

BACKGROUND: Furaltadone (FTD) is a type of nitrofuran and has been banned in many countries as a veterinary drug in food-producing animals owing to its potential carcinogenicity and mutagenicity. FTD is unstable in vivo, rapidly metabolizing to 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ); thus AMOZ can be used as an indicator for illegal usage of FTD. Usually, for the determination of nitrofurans, the analyte is often a derivative of the metabolite rather than the metabolite itself. In this study, based on the monoclonal antibody (mAb) against AMOZ, a competitive immunochromatographic assay (ICA) using a colloidal gold-mAb probe for rapid and direct detection of AMOZ without a derivatization step in meat and feed samples was developed. RESULTS: The intensity of red color in the test line is inversely related to the analyte concentration and the visual detection limit was found to be 10 ng mL⁻¹. The performance of this assay was simple and convenient because the tedious and time-consuming derivatization step was avoided. The ICA detection was completed within 10 min. The ICA strips could be used for 7 weeks at room temperature without significant loss of activity. The AMOZ spiked samples were detected by ICA and confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good agreement. CONCLUSION: The proposed ICA provides a feasible tool for simple, sensitive, rapid, convenient and semi-quantitative detection of AMOZ in meat and feed samples on site. To our knowledge, this is the first report of the ICA for direct detection of AMOZ.

[Accumulation law of epigoitrin in roots of Isatis indigotica of different breed types].

OBJECTIVE: To study the distribution law of epigoitrin in roots of Isatis indigotica of different breed types and provide a scientific basis for screening of high-quality Isatis indigotica breed. METHODS: Determined the contents of epigoitrin in tap root and lateral root of Chinese-cabbage-leaf Isatis, cabbage-leaf Isatis, mustard-leaf Isatis and tetraploid Isatis by HPLC. Also, compared the contents of epigoitin in xylem and phloem of Isatis indigotica. RESULTS: Contents of epigoitrin in the tap root and lateral root of Isatis indigotica of the different breed types were significant different. In four breed types of Isatis indigotica, contents of epigoitrin in the tap root and phloem were higher than those in the lateral root and xylem, respectively. CONCLUSION: Contents of epigoitrin in the lateral root of Isatis indigotica are higher than those of tap root and epigoitrin distributes mainly in phloem.

Mitochondrial cardiolipin is required for Nlrp3 inflammasome activation.

Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic linezolid as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. Cardiolipin bound to Nlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli.

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.

Synthesis and in vitro antibacterial activity of novel 3-azabicyclo[3.3.0]octanyl oxazolidinones.

We synthesized a series of oxazolidinone-type antibacterials in which morpholine C-ring of linezolid has been modified by substituted 3-azabicyclo[3.3.0]octanyl rings. Acetamide or 1,2,3-triazole heterocycle was used as C-5 side chain of oxazolidinone. The resulting series of compounds was then screened in vitro against panel of susceptible and resistant Gram-positive, Gram-negative bacteria, and Mycobacterium tuberculosis (Mtb). Several analogs in this series exhibited potent in vitro antibacterial activity comparable or superior to linezolid against the tested bacteria. Compounds 10a, 10b, 11a, and 15a displayed highly potent activity against M. tuberculosis. Selected compound 10b showed good human microsomal stability and CYP-profile, and showed low activity against hERG channel.

Detection, accumulation, distribution, and depletion of furaltadone and nifursol residues in poultry muscle, liver, and gizzard.

Nitrofurans were broadly used as an extremely effective veterinary antibiotic especially in pig and poultry production farms. Because of fears of the carcinogenic effects on humans, the nitrofurans were banned from use in livestock production in many countries, including the European Union. The present study examines the accumulation, distribution, and depletion of furaltadone and nifursol and of their tissue-bound metabolites [3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and 3,5-dinitro-salicylic acid hydrazine (DNSAH), respectively, in poultry edible tissues (muscle, liver, and gizzards) following administration to chickens of therapeutic and subtherapeutic concentrations of both compounds. Nitrofurans determination was performed by high-performance liquid chromatography-diode array detection and liquid chromatography-tandem mass spectrometry, respectively, for feeds and for poultry tissues. Furaltadone and nifursol, in very low concentrations, were found in samples of muscle, liver, and chicken's gizzard collected from slaughtered animals after 5 weeks of treatment and no withdrawal time period. When a withdrawal time period of 3 weeks was respected, no detectable nitrofuran parent compounds was observed in all of the studied matrices. For AMOZ, concentrations of 270 µg/kg in meat, 80 µg/kg in liver, and 331 µg/kg in gizzard were determined after administration of a medicated feed with furaltadone (132 mg/kg), 3 weeks after withdrawal of treatment. For DNSAH, the concentration values obtained are much lower than those observed for AMOZ. For meat, liver, and gizzard, DNSAH concentrations of 2.5, 6.4, and 10.3 µg/kg, respectively, were determined, after administration of a medicated feed with nifursol (98 mg/kg), 3 weeks after withdrawal of treatment. The gizzard could be considered a selected matrix for nitrofuran residues evaluation in poultry, due to its capacity of retaining either nitrofuran parent compounds or metabolites in higher concentrations, regardless of the administered dose or of the respected withdrawal time period.

Preclinical and clinical evidence for the collaborative transport and renal secretion of an oxazolidinone antibiotic by organic anion transporter 3 (OAT3/SLC22A8) and multidrug and toxin extrusion protein 1 (MATE1/SLC47A1).

N-({(5S)-3-[4-(1,1-dioxidothiomorpholin-4-yl)-3,5-difluorophenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide (PNU-288034), an oxazolidinone antibiotic, was terminated in phase I clinical development because of insufficient exposure. Analysis of the drug pharmacokinetic and elimination profiles suggested that PNU-288034 undergoes extensive renal secretion in humans. The compound was well absorbed and exhibited approximately linear pharmacokinetics in the oral dose range of 100 to 1000 mg in human. PNU-288034 was metabolically stable in liver microsomes across species, and unchanged drug was cleared in the urine by an apparent active renal secretion process in rat and monkey (two to four times glomerular filtration rate) but not dog. In vitro studies conducted to characterize the transporters involved demonstrated PNU-288034 uptake by human organic anion transporter 3 (OAT3; K(m) = 44 +/- 5 microM) and human multidrug and toxin extrusion protein 1 (hMATE1; K(m) = 340 +/- 55 microM). The compound was also transported by multidrug resistance P-glycoprotein and breast cancer resistance protein. In contrast, human organic cation transporter 2, human OAT1, and hMATE2-K did not transport PNU-288034. Coadministration of PNU-288034 and the OAT3 inhibitor probenecid significantly increased PNU-288034 plasma area under the curve (170%) and reduced both plasma and renal clearance in monkey. Coadministration of PNU-288034 and cimetidine, a MATE1 inhibitor, also reduced plasma clearance in rat to a rate comparable with probenecid coadministration. Collectively, our results demonstrated a strong in vitro-in vivo correlation for active renal secretion coordinated through the vectorial transport process of OAT3 and MATE1, which ultimately resulted in limiting the systemic exposure of PNU-288034.

Application of affinity selection/mass spectrometry to determine the structural isomer of parnafungins responsible for binding polyadenosine polymerase.

To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).

Synthesis of poly(ethylene glycol)-metaxalone conjugates and study of its controlled release in vitro.

Metaxalone (Met), a drug for treatment of pain and stiffness due to muscular injuries, was covalently linked to poly(ethylene glycols) (PEG) via a chloroacetyl chloride spacer. The average weight molecular weights used for PEG are 4000, 6000 and 10,000, respectively, and the procedure of chemical modification for PEGs was conducted by a two-step protocol: (1) synthesis of N-chloroacetyl-metaxalone; (2) synthesis of PEG(4000)-Met, PEG(6000)-Met and PEG(10000)-Met. The controlled drug release studies were performed in buffer solutions with pH values equal to 1.1, 7.4 and 10.0. The results demonstrate that, in the same condition, the rate of hydrolysis for PEG(10000)-Met is the slowest among three prodrugs, and more amount of metaxalone can be detected releasing from prodrug matrices at the presence of alpha-chymotrypsin in a buffer solution with pH 8.0. It was also found that these novel prodrugs can effectively improve the metaxalone's pharmacokinetics, and furthermore can markedly increase its half-life period.

Intrapulmonary penetration of linezolid.

OBJECTIVE: This study was designed to measure the concentrations of linezolid in bronchial mucosa, pulmonary macrophages and epithelial lining fluid and to compare them with simultaneous blood levels. METHODS: Ten adult patients undergoing bronchoscopy for diagnostic purposes were given oral linezolid at a dosage of 600 mg twice a day for a total of six doses. Patients with active lung infection were excluded from the study. Flexible bronchoscopy was carried out between 2 and 8 h after the last dose of linezolid. Bronchial biopsies and bronchoalveolar lavage were carried out and a simultaneous blood sample obtained. Linezolid levels were measured using high-performance liquid chromatography (HPLC). RESULTS: Mean concentrations of linezolid were 13.4 mg/L in serum, 10.7 mg/kg in mucosa, 8.1 mg/L in alveolar macrophages and 25.1 mg/L in epithelial lining fluid. The mean site/serum concentration ratios were 0.79 for bronchial mucosa, 0.71 for macrophages and 8.35 for epithelial lining fluid. CONCLUSIONS: The MIC90 (< or =4 mg/L) of linezolid for Staphylococcus aureus and Streptococcus pneumoniae was exceeded in serum and bronchial mucosa in all subjects, in epithelial lining fluid in nine subjects and in macrophages in six subjects.

Catalytic properties of mutant 23 S ribosomes resistant to oxazolidinones.

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 Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki 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 substrate-independent 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.