Development of a fluorescence-based enzyme assay of human 5-lipoxygenase.
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.
Pufahl
Robert A
RA
Pfizer Global Research and Development, Chesterfield, MO 63017, USA. robert.a.pufahl@pfizer.com
Kasten
Thomas P
TP
Hills
Rob
R
Gierse
James K
JK
Reitz
Beverly A
BA
Weinberg
Robin A
RA
Masferrer
Jaime L
JL
D001094:Arachidonate 5-Lipoxygenase / Q000302:isolation & purification*; D002863:Chromogenic Compounds / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D020128:Inhibitory Concentration 50; D017572:Leukotriene A4 / Q000737:chemistry; D015289:Leukotrienes / Q000737:chemistry; D016859:Lipoxygenase Inhibitors / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D017572:Leukotriene A4; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; C029569:diacetyldichlorofluorescein; C025348:arachidonic acid 5-hydroperoxide; D001094:Arachidonate 5-Lipoxygenase