Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe

Small-molecule inhibitor of OGG1 suppresses pro-inflammatory gene expression and inflammation

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]+ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MSn analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well

Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons

Formation of DNA adducts as a result of exposure to polycyclic aromatic hydrocarbons (PAH) was studied in 98 potroom workers from an aluminium smelting plant and in 55 blue-collar workers without occupational PAH exposure. DNA from peripheral blood mononuclear cells (PBMC) was used for quantitation of individual PAH-DNA adducts by P-32-postlabelling/high performance liquid chromatography (HPLC) analysis. Four individual DNA adducts (denoted A, B, C and D) were quantified in 141 of a total of 153 subjects. Genetic polymorphisms for cytochrome P-4501A1 (CYP1A1), Microsomal epoxide hydrolase, N-acetyltransferase 2, glutathione transferases M1, P1 and T1 (GSTM1, GSTP1 and GSTT1, respectively) and NAD(P)H: quinone oxidoreductase I (NQ01) were analysed. For 52 subjects, analysis of mRNA inducibility of CYP1A1 was performed. No statistically significant differences in the levels of total or individual DNA adducts A, C and D were found between potroom workers and control subjects. All potroom workers and the subgroup of potroom workers who reported to never/sometimes use personal respiratory protection (n = 72) were found to have a significantly higher likelihood of having high levels of adduct B than control subjects [odds ratio (OR) = 3.4 with 95% confidence interval (CI) of 1.3-9.2, and OR=4.2 with 95% CI 1.6-11.5, respectively]. In the subgroup, levels of adducts A and B were found to be significantly higher among workers with employment time of less than 6 months (n = 5). Also, the levels of the individual DNA adducts were to some extent modified by genetic polymorphisms in CYP1A1, GSTM1, GSTP1 and NQO1 and by CYP1A1 inducibility. In conclusion, levels of adduct B, identified by P-32-postlabelling/HPLC methodology as an indicator of PAH exposure in aluminium production, were modified by the use of respiratory protection, length of employment and genetic polymorphisms