Application of the ferrous oxidation-xylenol orange assay for the screening of 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LO) is the key enzyme involved in leukotriene synthesis and its improper regulation is implicated in several inflammatory diseases. A rapid and sensitive assay for 5-LO activity suitable for high-throughput format is not yet available. In this study, we examined whether the ferrous oxidation-xylenol orange (FOX) assay could be applicable for the high-throughput screening of 5-LO inhibitors. Using insect cell lysates overexpressing rat 5-LO, the effects of cofactors of 5-LO such as ATP, Ca2+, and L-alpha-phosphatidylcholine (PC) on the color development of FOX reagents were investigated. ATP quenched substantially color development by hydroperoxide, an intermediate of 5-LO reaction, and an Optimum concentration of ATP with little interference was determined as 20 PM. Ethylenediaminetetraacetate (0.4mM) also affected the complex formation with FOX reagents. On the other hand, neither Ca2+ nor PC influenced complex formation with FOX reagents. Under optimized assay conditions, zileuton, a 5-LO-specific inhibitor.. exhibited inhibitory potency (IC50 values of 0.1-0.2 mu M) similar to that determined by the conventional spectrophotometric assay. Taken together, this study shows that the FOX assay with some modifications can be employed for high-throughput assay format for the measurement of 5-LO activity at the stage of primary screening. (c) 2005 Elsevier Inc. All rights reserved.