Journal
ANALYTICAL BIOCHEMISTRY
Volume 364, Issue 2, Pages 204-212Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.02.009
Keywords
5-lipoxygenase; 2 ',7 '-dichlorodihydrofluorescein; arachidonic acid; hydroperoxide; high-throughput screen
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Leukotrienes are important mediators in a number of inflammatory diseases and therefore are a target of several therapeutic approaches. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotriene A(4) (LTA(4)) in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). Assays to measure 5-LOX activity typically have been low throughput and time consuming. In this article, we describe a fluorescence assay that is amenable to high-throughput screening in a 384-well microplate format. The fluorescent signal is measured during oxidation of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by human 5-LOX. The assay has been found to reliably identify small molecule inhibitors of human 5-LOX. The IC50 values of several 5-LOX inhibitors in this new assay are comparable to those determined in a standard spectrophotometric assay that measures the formation of the 5(S)-hydroperoxyeicosatetraenoic acid (5.-HpETE) product. In addition, we demonstrate the use of the assay in a high-throughput screen of the Pfizer compound collection to identify inhibitors of 5-LOX. (c) 2007 Elsevier Inc. All rights reserved.
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