TY  - JOUR
A1  - Meyners, Christian
A1  - Wawrzinek, Robert
A1  - Kraemer, Andreas
A1  - Hinz, Steffen
A1  - Wessig, Pablo
A1  - Meyer-Almes, Franz-Josef
T1  - A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe
JF  - Analytical & bioanalytical chemistry
N2  - High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3]benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N'-phenyloctandiamide.
KW  - Histone deacetylases
KW  - Acetylpolyamine amidohydrolases
KW  - Fluorescence life time
KW  - Binding assay
KW  - Pseudomonas aeruginosa
Y1  - 2014
U6  - https://doi.org/10.1007/s00216-014-7886-5
SN  - 1618-2642
SN  - 1618-2650
VL  - 406
IS  - 20
SP  - 4889
EP  - 4897
PB  - Springer
CY  - Heidelberg
ER  - 
TY  - JOUR
A1  - Pewzner-Jung, Yael
A1  - Tabazavareh, Shaghayegh Tavakoli
A1  - Grassme, Heike
A1  - Becker, Katrin Anne
A1  - Japtok, Lukasz
A1  - Steinmann, Joerg
A1  - Joseph, Tammar
A1  - Lang, Stephan
A1  - Tuemmler, Burkhard
A1  - Schuchman, Edward H.
A1  - Lentsch, Alex B.
A1  - Kleuser, Burkhard
A1  - Edwards, Michael J.
A1  - Futerman, Anthony H.
A1  - Gulbins, Erich
T1  - Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa
JF  - EMBO molecular medicine
N2  - Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P.aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P.aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.
KW  - cystic fibrosis
KW  - long chain base
KW  - lung infection
KW  - Pseudomonas aeruginosa
KW  - sphingosine
Y1  - 2014
U6  - https://doi.org/10.15252/emmm.201404075
SN  - 1757-4676
SN  - 1757-4684
VL  - 6
IS  - 9
SP  - 1205
EP  - 1214
PB  - Wiley-Blackwell
CY  - Hoboken
ER  -