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Journal of Biomolecular Screening
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Development of a Homogeneous Time-Resolved Fluorescence Leukotriene B4 Assay for Determining the Activity of Leukotriene A4 Hydrolase

Amy M. Liang

Molecular Pharmacology, Berlex Biosciences, Richmond, California, amy_liang{at}berlex.com

Emmanuel Claret

HTRF R&D Department, Cisbio International, BP 84175, 30204 Bagnols sur Cèze Cedex, France

Josy Ouled-Diaf

HTRF R&D Department, Cisbio International, BP 84175, 30204 Bagnols sur Cèze Cedex, France

Alexandre Jean

HTRF R&D Department, Cisbio International, BP 84175, 30204 Bagnols sur Cèze Cedex, France

David Vogel

Antibody Technology, Berlex Biosciences. Richmond, California

David R. Light

Antibody Technology, Berlex Biosciences. Richmond, California

Steven W. Jones

Molecular Pharmacology, Berlex Biosciences, Richmond, California

William J. Guilford

Medicinal Chemistry, Berlex Biosciences, Richmond, California

John F. Parkinson

Immunology, Berlex Biosciences, Richmond, California

R. Michael Snider

Molecular Pharmacology, Berlex Biosciences, Richmond, California

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 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 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 LTB 4. 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)

Key Words: leukotriene B4 • HTRF assay • leukotriene A4 hydrolase • leukotriene A4 hydrolase inhibitor • inhibitor potency

This version was published on June 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 4, 536-545 (2007)
DOI: 10.1177/1087057107299873
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