Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase.
Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic 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 suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.
Liang
Amy M
AM
Molecular Pharmacology, Berlex Biosciences, Richmond, California 94804, USA. amy_liang@berlex.com
Claret
Emmanuel
E
Ouled-Diaf
Josy
J
Jean
Alexandre
A
Vogel
David
D
Light
David R
DR
Jones
Steven W
SW
Guilford
William J
WJ
Parkinson
John F
JF
Snider
R Michael
RM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D004789:Enzyme Activation; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D015200:Fluoroimmunoassay; D006801:Humans; D007975:Leukotriene B4 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D007975:Leukotriene B4; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase