Development of a fluorescence-based enzyme assay of human 5-lipoxygenase

• DOI: 10.1016/j.ab.2007.02.009
• Publication Date: 2007-02-15T07:04:31Z
• Authors: Robert A. Pufahl; Thomas P. Kasten; Rob Hills; James K. Gierse; Beverly A. Reitz; Robin A. Weinberg; Jaime L. Masferrer
• Keywords: High-Throughput Screening; Plate reader; Leukotriene B4; IC50

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 IC(50) 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.