Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A(4) (LTA(4)) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B-4 (LTB4), a potent lipid chemotatic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA(4) hydrolase is regarded as an attractive therapeutic target for anti- i nfl animation. To facilitate identification and optimization of LTA(4) hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB, (LTB4 HTRF (R) assay) and its application to establish an HTRF-based LTA, hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA(4) hydrolase reaction, an HTRFbased LTA(4) hydrolase assay was established and validated. Using a test set of 16 LTA(4) hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB4 enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA(4) hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA(4) hydrolase.