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Fluorescent leukotriene B-4: potential applications

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Author

  • A Sabirsh
  • A Wetterholm
  • Jesper Bristulf
  • Hakon Leffler
  • JZ Haeggstrom
  • Christer Owman

Summary, in English

Leukotriene B-4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a K-d of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Publishing year

2005

Language

English

Pages

1339-1346

Publication/Series

Journal of Lipid Research

Volume

46

Issue

6

Document type

Journal article

Publisher

American Society for Biochemistry and Molecular Biology

Topic

  • Neurosciences
  • Microbiology in the medical area
  • Immunology in the medical area

Keywords

  • G-protein coupled receptor
  • pharmacology
  • method

Status

Published

Research group

  • Drug Target Discovery

ISBN/ISSN/Other

  • ISSN: 1539-7262