5-lipoxegynase (5-LOX) Redistribution Assay

• Designed to assay compounds for their ability to modulate translocation of 5-LOX to the nuclear envelope
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

5-Lipoxygenase (5-LOX) is a lipid-peroxidizing enzyme that plays an essential role in the biosynthesis of leukotrienes, which mediate inflammatory and allergic reactions. The key regulatory steps in leukotriene biosynthesis following cell activation include Ca²⁺ mobilization and subsequent release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2. In a Ca^2+- and ATP-dependent reaction, AA is then metabolized by 5-LOX to yield the epoxide intermediate leukotriene A4 (LTA4). This step is dependent upon the interaction of 5-LOX with the nuclear membrane protein 5-lipoxygenase activating protein (FLAP) and requires translocation of 5-LOX to the nuclear envelope. FLAP then presents AA to 5-LOX and thereby increases the catalytic potential of 5-LOX. In addition to being activated by an increase in intracellular Ca²⁺ concentration, 5-LOX can be activated by diacylglycerols as well as by phosphorylation by MAPKAP kinase-2 and ERK.

In addition to the classical application of leukotriene synthesis inhibitors in asthma and allergic disorders, leukotriene synthesis inhibitors might be of value for the treatment of cardiovascular diseases and osteoporosis. Moreover, dual 5-LOX/COX inhibitors are potential new drugs to treat inflammation, since they act by blocking the formation of both prostaglandins and leukotrienes.

Figure 1. Translocation of 5-LOX-EGFP to the nuclear envelope. Cells were treated with 300 nM A23187 for 5 min (right panel) or untreated (DMSO control, left panel). Arrows indicate the nuclear envelope localization detected by the image analysis algorithm.

Figure 2. Concentration response curves in the 5-LOX assay: A) A23187 concentration response. The EC₅₀ is approximately 35 nM. Concentration response was measured in 9 point half log dilution series (n=16). Cells were treated with A23187 for 5 min. Cells were then fixed and translocation to the nuclear envelope was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (300 nM A23187) and negative control (0.25% DMSO). B) Ionomycin concentration response. The EC₅₀ is approximately 30 nM. Concentration response was measured in 7 point half log dilution series (n= 16). Cell treatment and image analysis was performed as in A).