title: Characterization of a 5-lipoxygenase-activating protein binding assay: correlation of affinity for 5-lipoxygenase-activating protein with leukotriene synthesis inhibition.
abstract: A binding assay has been developed to measure the affinity of leukotriene synthesis inhibitors for 5-lipoxygenase-activating protein (FLAP), using human leukocyte membranes as the source of FLAP and a radioiodinated leukotriene synthesis inhibitor, 125I-L-691,831, as ligand. Linearity of specific binding of radiolabeled ligand was demonstrated with increasing protein and ligand concentrations. Saturation analysis of radioligand binding showed a Kd of 6 nM and a Bmax that, depending on the membrane preparation, varied between 8 and 53 pmol/mg of protein. An excellent correlation was shown between affinity for FLAP in the binding assay and inhibition of leukotriene synthesis in human polymorphonuclear leukocytes for compounds from two structurally distinct classes, namely indoles and quinolines. A large number of membrane-active compounds did not compete with 125I-L-691,831 binding to FLAP. In addition, direct 5-lipoxygenase inhibitors and a selection of eicosanoids were unable to compete for FLAP binding. This study validates a selective binding assay for leukotriene synthesis inhibitors whose protein target is FLAP.
authors.affiliation: Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Quebec, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Charleson
authors.forename: P
authors.initials: P
authors.lastname: Prasit
authors.forename: S
authors.initials: S
authors.lastname: Léger
authors.forename: J W
authors.initials: JW
authors.lastname: Gillard
authors.forename: P J
authors.initials: PJ
authors.lastname: Vickers
authors.forename: J A
authors.initials: JA
authors.lastname: Mancini
authors.forename: P
authors.initials: P
authors.lastname: Charleson
authors.forename: J
authors.initials: J
authors.lastname: Guay
authors.forename: A W
authors.initials: AW
authors.lastname: Ford-Hutchinson
authors.forename: J F
authors.initials: JF
authors.lastname: Evans
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D011485:Protein Binding; D011804:Quinolines / Q000378:metabolism*; D013189:SRS-A / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D007211:Indoles; D016859:Lipoxygenase Inhibitors; D008565:Membrane Proteins; D011804:Quinolines; D013189:SRS-A; C074616:L 691831