Development of a Homogeneous Time-Resolved Fluorescence Leukotriene B 4 Assay for Determining the Activity of Leukotriene A 4 Hydrolase

Abstract

Leukotriene A₄ (LTA₄) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B₄ (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 LTA₄ hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA₄ 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 LTB₄ (LTB₄ HTRF^® assay) and its application to establish an HTRF-based LTA₄ 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 LTA₄ hydrolase reaction, an HTRF-based LTA₄ hydrolase assay was established and validated. Using a test set of 16 LTA₄ hydrolase inhibitors, a good correlation was found between the IC₅₀ values obtained using LTB₄ HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA₄ hydrolase assay was shown to be an efficient and suitable⁴ assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA₄ hydrolase. (Journal of Biomolecular Screening 2007:536-545)

Keywords

leukotriene B4 HTRF assay leukotriene A4 hydrolase leukotriene A4 hydrolase inhibitor inhibitor potency

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