title: Leukotriene B₄ Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B₄ In Vitro and In Cellulo.
abstract: LTB4 is an inflammatory lipid mediator mainly biosynthesized by leukocytes. Since its implication in inflammatory diseases is well recognized, many tools to regulate its biosynthesis have been developed and showed promising results in vitro and in vivo, but mixed results in clinical trials. Recently, the mTOR pathway component p70S6 kinase 1 (p70S6K1) has been linked to LTC4 synthase and the biosynthesis of cysteinyl-leukotrienes. In this respect, we investigated if p70S6K1 could also play a role in LTB4 biosynthesis. We thus evaluated the impact of the p70S6K1 inhibitors PF-4708671 and LY2584702 on LTB4 biosynthesis in human neutrophils. At a concentration of 10 μM, both compounds inhibited S6 phosphorylation, although neither one inhibited the thapsigargin-induced LTB4 biosynthesis, as assessed by the sum of LTB4, 20-OH-LTB4, and 20-COOH-LTB4. However, PF-4708671, but not LY2584702, inhibited the ω-oxidation of LTB4 into 20-OH-LTB4 by intact neutrophils and by recombinant CYP4F3A, leading to increased LTB4 levels. This was true for both endogenously biosynthesized and exogenously added LTB4. In contrast to that of 17-octadecynoic acid, the inhibitory effect of PF-4708671 was easily removed by washing the neutrophils, indicating that PF-4708671 was a reversible CYP4F3A inhibitor. At optimal concentration, PF-4708671 increased the half-life of LTB4 in our neutrophil suspensions by 7.5 fold, compared to 5 fold for 17-octadecynoic acid. Finally, Michaelis-Menten and Lineweaver-Burk plots indicate that PF-4708671 is a mixed inhibitor of CYP4F3A. In conclusion, we show that PF-4708671 inhibits CYP4F3A and prevents the ω-oxidation of LTB4 in cellulo, which might result in increased LTB4 levels in vivo.
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.|Département de médecine, Faculté de médecine, Université Laval, Québec City, Québec, Canada.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Archambault
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.|Département de médecine, Faculté de médecine, Université Laval, Québec City, Québec, Canada.
authors.forename: Caroline
authors.initials: C
authors.lastname: Turcotte
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.
authors.forename: Cyril
authors.initials: C
authors.lastname: Martin
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.
authors.forename: Julie S
authors.initials: JS
authors.lastname: Lefebvre
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.
authors.forename: Véronique
authors.initials: V
authors.lastname: Provost
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.|Département de médecine, Faculté de médecine, Université Laval, Québec City, Québec, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Laviolette
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada.|Département de médecine, Faculté de médecine, Université Laval, Québec City, Québec, Canada.
authors.forename: Nicolas
authors.identifier: http://orcid.org/0000-0001-5944-7200
authors.initials: N
authors.lastname: Flamand
mesh_terms: D000072470:Cytochrome P450 Family 4 / Q000037:antagonists & inhibitors*; D004789:Enzyme Activation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007975:Leukotriene B4 / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects*; D010879:Piperazines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D007093:Imidazoles; C552719:PF-4708671; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D007975:Leukotriene B4; D000072470:Cytochrome P450 Family 4; C465349:CYP4F3 protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa