In vitro and in vivo activities of novel, semisynthetic thiopeptide inhibitors of bacterial elongation factor Tu.

Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the in vitro and in vivo activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC(90) ≤ 0.25 µg/ml) but weaker against the streptococci (MIC(90) ≥ 4 µg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the tuf gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal S. aureus infections with 50% effective doses (ED(50)) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic E. faecalis infections with ED(50) of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active in vitro and in vivo activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.

A novel HLA-DRB1*13 allele (DRB1*1357) identified by polymerase chain reaction with sequence-specific primers and direct sequencing.

We describe the identification of a new DRB1*13 allele, DRB1*1357*, found in two Austrian Caucasian individuals. The novel allele was initially suspected because analysis with sequence-specific primers resulted in an unusual pattern of amplification. Thereafter, exon 2 was further characterized by sequence-based typing. The nucleotide sequence of DRB1*1357 is identical to DRB1*1319 except for a single substitution in codon 47 (TAC to TTC) leading to a change from phenylalanine to tyrosine.

Detection of in vivo formed DNA adducts at the part-per-billion level by capillary liquid chromatography/microelectrospray mass spectrometry.

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vivo sources. Adjustments were made to a previously described methodology such that analyte detection could be improved by nearly 2 orders of magnitude. These adjustments included changing the electrospray ionization sprayer configuration, increasing the sample injection volume, improving the solid-phase extraction procedure, and increasing peak efficiency by modifying chromatographic conditions. While this scheme for improving analyte detection was targeted for DNA adducts, it could be applied to almost any LC/MS methodology where sensitive analysis is the primary objective. Selective reaction monitoring) techniques with a triple quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts, with detection limits approaching 1 adduct in 10(9) unmodified bases using approximately 500 microg of DNA. The DNA adducts N-(2'-deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline and 5-(2'-deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline were detected in pancreas tissue of a cynomolgus monkey sacrificed 24 h after a single administration of 10 mg/kg carcinogen. The LC/MS results were consistent with previously published 32P-postlabeling data (Turesky et al. Chem Res. Toxicol. 1996, 9, 403-408). Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

Reduction of signal suppression effects in ESI-MS using a nanosplitting device.

Electrospray ionization mass spectrometry is a valuable tool in the identification and quantification of drug metabolites in biological fluids. However, there are many instances where matrix components present in these fluids interfere with analyte detection and prevent the acquisition of accurate or complete results. In some instances, the matrix can suppress ionization to such an extent that analytes are completely undetectable by MS. In this work, we investigate how ionization and ion-transfer efficiencies are affected by drastically reducing the flow into the MS. A postcolumn concentric flow-splitting device was constructed to allow the measurement of analyte signal and ionization suppression across a range of flow rates (0.1-200 microL/min). Using this device, the effects of flow rate on signal intensity and ionization suppression were measured in analytical experiments that included flow injection analysis MS, postcolumn addition LC-MS, and on-line LC-MS analysis of metabolites generated from rat liver microsomes. The device used to deliver 0.1 microL/min flows is referred to as a nanosplitter because it achieved high split ratios (2000:1), producing flow rates comparable to those observed in nanoelectrospray. The nanosplitter maintained chromatographic integrity with high fidelity and allowed the direct comparison of analyte signal across a range of flow rates (0.1-200 microL/min). A significant improvement in concentration and mass sensitivity as well as a reduction in signal suppression is observed when the performance at 200 versus 0.1 microL/min flow rate is compared. Using this specially designed concentric splitting device, the advantages of ultralow flow ESI were easily exploited for applications employing large bore chromatography.

Subatmospheric electrospray interface for coupling of microcolumn separations with mass spectrometry.

A modular subatmospheric electrospray interface with fiber optic UV detection close to the electrospray tip was developed for coupling of microcolumn separation techniques with mass spectrometry. The interface was based on a liquid junction with a removable microelectrospray tip. The electrospray tip was enclosed in a subatmospheric chamber attached in front of the sampling orifice of the mass spectrometer. The inlet of the liquid junction was maintained at atmospheric pressure, and thus no pressure drop developed across the separation column. The flow rate of the electrosprayed liquid from the liquid junction reservoir was adjusted by the pressure in the electrospray chamber. In this approach, a continuous and stable electrospray could be achieved without the use of an external pump. Since the electrospray did not depend on fluid delivery from the separation column, coated capillaries without electroosmotic flow as well as capillaries with electroosmotic flow could be used for capillary electrophoresis. In addition, the interface was found to be effective with capillary liquid chromatography. The use of a fiber optic UV detector placed close to the exit of the separation column provided additional detection information and a simple means of troubleshooting. The interface did not significantly influence the quality of the separation, even with columns generating several hundred thousand theoretical plates. Peptide samples in the submicromolar concentration range were detected, corresponding to a limit of detection in the attomole range.

Determination of in vitro- and in vivo-formed DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline by capillary liquid chromatography/microelectrospray mass spectrometry.

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vitro and in vivo sources. Constant neutral loss (CNL) and selective reaction monitoring (SRM) techniques with a triple-quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts in vitro and in animals. Detection of 1 adduct in 10(4) unmodified bases is achieved using CNL scanning detection, while the lower detection limits using SRM approach 1 adduct in 10(7) unmodified bases using 300 microg of DNA. The DNA adducts N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4, 5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N(2)-IQ) were detected in kidney tissues of chronically treated cynomolgus monkeys at levels and in proportions consistent with previously published (32)P-postlabeling data [Turesky, R. J., et al. (1996) Chem. Res. Toxicol. 9, 403-408]. Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human intestinal microsomes and expressed cytochrome P4503A4/3A5: mechanism-based inactivation of cytochrome P4503A by ritonavir.

Both ritonavir and indinavir were readily metabolized by human intestinal microsomes. Comparison of the patterns of metabolites in incubations with enterocyte microsomes and expressed cytochrome P450 (CYP) isozymes and immunoinhibition and chemical inhibition studies showed the essential role of the CYP3A subfamily in the metabolism of both protease inhibitors by the small intestine. Ritonavir was similarly biotransformed by microsomes containing expressed CYP3A4 or CYP3A5 isozymes (KM = 0.05-0.07 microM, Vmax = 1-1.4 nmol/min/nmol CYP). In contrast, both the patterns of metabolites and the enzyme kinetic parameters for the metabolism of indinavir by expressed CYP3A5 (KM = 0.21 microM, Vmax = 0.24 nmol/min/nmol CYP) and CYP3A4 (KM = 0.04 microM, Vmax = 0.68 nmol/min/nmol CYP) were different. The biotransformation of both indinavir and ritonavir in human enterocyte microsomes was characterized by very low KM values (0.2-0.4 microM for indinavir and <0.1 microM for ritonavir). The Vmax for indinavir metabolism was greater in enterocyte (163 +/- 35 pmol/min/mg protein) than in liver (68 +/- 44 pmol/min/mg protein) microsomes. The metabolism of ritonavir in liver and enterocyte microsomes was associated with inactivation of CYP3A. The initial Vmax for ritonavir metabolism by enterocyte microsomes was 89 +/- 59 pmol/min/mg protein. The apparent inactivation rate constants for intestinal CYP3A and expressed CYP3A4 were 0.078 and 0.135 min-1, respectively. Metabolic inactivation of CYP3A by ritonavir explains the improved bioavailability and pharmacokinetics of ritonavir and the sustained elevation of blood levels of other, concomitantly administered, substrates of CYP3A.