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
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  • 作者:Christian Meyners (1)
    Robert Wawrzinek (2)
    Andreas Kr?mer (1)
    Steffen Hinz (1)
    Pablo Wessig (2)
    Franz-Josef Meyer-Almes (1)
  • 关键词:Histone deacetylases ; Acetylpolyamine amidohydrolases ; Fluorescence life time ; Binding assay ; Pseudomonas aeruginosa
  • 刊名:Analytical and Bioanalytical Chemistry
  • 出版年:2014
  • 出版时间:August 2014
  • 年:2014
  • 卷:406
  • 期:20
  • 页码:4889-4897
  • 全文大小:369 KB
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  • 作者单位:Christian Meyners (1)
    Robert Wawrzinek (2)
    Andreas Kr?mer (1)
    Steffen Hinz (1)
    Pablo Wessig (2)
    Franz-Josef Meyer-Almes (1)

    1. Department of Chemical Engineering and Biotechnology, University of Applied Sciences Darmstadt, Schnittspahnstr. 12, 64287, Darmstadt, Germany
    2. Institut für Chemie, Universit?t Potsdam, 14476, Potsdam, Germany
  • ISSN:1618-2650
文摘
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. Figure ?/em>
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