br Acknowledgments This work was supported by

Acknowledgments This work was supported by the Swiss National Science Foundation.
15-Lipoxygenase 1 (15-LOX-1) is a nonheme, iron-containing enzyme predominantly expressed in reticulocytes, eosinophils, macrophages, mast cells, and bronchial epithelial cells . 15-LOX-1 is a key enzyme involved in fatty AMG 9810 sale metabolism that oxidizes unsaturated fatty acids, including arachidonic acid; preferentially generates active 15()-hydroperoxy-5(),8(),11(),13()-eicosatetraenoic acid (15-HpETE); and produces small amounts of 12()-hydroperoxy-5(),8(),10(),14()-eicosatetraenoic acid (12-HpETE) , . 15-LOX-1 has been reported to participate in various pathophysiological processes including cell proliferation, differentiation, apoptosis, and angiogenesis , , . The biological and medical research on 15-LOX-1 in humans has mainly been focused on its potential role in chronic inflammatory airway diseases including asthma and chronic obstructive pulmonary disease , . Increased expression and activity of 15-LOX-1 have been demonstrated in the respiratory tissue of asthma patients, suggesting a pathophysiological role for this enzyme in chronic airway inflammation . All the above studies suggest that 15-LOX-1 is a promising target for chronic inflammation and highlight the fact that developing specific and potent 15-LOX-1 inhibitors is extremely important for both research and clinical interests. To date, a number of potent 15-LOX-1 inhibitors derived from various sources have been reported , , , , , , , . The most promising 15-LOX-1 inhibitors so far are the tryptamine-, imidazole-, and 1,3,4-oxadiazole-based derivatives, which have low-nanomolar inhibitory activity and selectivity against both 5-lipoxygenase and 12-lipoxygenase , , . However, none of the 15-LOX-1-specific inhibitors has been applicable in clinical use yet. Hence, there is a continually growing need to discover potent and selective 15-LOX-1 inhibitors for further development into therapeutic candidates for inflammation treatment. Although 15-LOX-1 has proved to be a promising inflammation therapeutic target, high-throughput enzymatic assays for 15-LOX-1 still are not well developed. Several analytical techniques for 15-LOX-1 have been reported, such as continuous spectrophotometric measurement at 236nm and determination of oxygen consumption rate , . However, these methods are too laborious and time consuming to quickly and effectively identify inhibitors of 15-LOX-1 and are not easily adaptable for high-throughput 15-LOX-1 inhibitor screening. In this study, we describe the development of a rapid and sensitive fluorimetric assay for the determination of human 15-LOX-1 activity. This assay utilizes the ability of 15-LOX-1-generated lipid hydroperoxides that oxidize nonfluorescent dihydrorhodamine 123 (DHR) to the highly fluorescent dye rhodamine 123 (A). DHR is a cell-permeative fluorogenic probe that is used as an indicator of the formation of the intracellular peroxide peroxynitrite peroxyl and hydroxyl radicals . However, there are few studies that have used DHR to detect the formation of lipid hydroperoxides. To our knowledge, this is the first use of DHR to determine the lipid hydroperoxides generated by 15-LOX-1. 15-LOX-1 derived from prokaryotic expression in and eukaryotic expression in HEK293T cells was used for this study. Human 15-LOX-1 was expressed as N-terminally His-tagged proteins in and purified by Ni-NTA column with an estimated purity of greater than 90% based on Coomassie-stained SDS–PAGE (). Human 15-LOX-1 in cell lysates was prepared by transfection of the expression plasmid pCDNA3-15-LOX-1 into HEK293T cells (see ). 15-LOX-1 fluorimetric assays were performed in white 96-well flat-bottom plates in a total assay volume of 100μl. Diluted recombinant human 15-LOX-1 (30nM) or cell lysates (10μg) in 90μl assay buffer (50mM Tris–HCl, pH 7.4) and 5μl DHR (final concentration 5μM) were mixed in an assay well. The enzymatic reaction was initiated by adding 5μl of arachidonic acid solution (1mM stock solution) to a final concentration of 50μM. The fluorescence was monitored over 20min at 2-min intervals at room temperature through a Synergy Multi-Mode microplate reader (BioTek, USA) using an excitation wavelength 500nm and an emission wavelength 536nm. To discover novel lead compounds from a chemical library, the development of a high-throughput screening method is essential. Therefore, the assay in 96-well microplate format was adapted to a 384-well microplate format by scaling down the reaction volume to 50μl.