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Open Access

Peer-reviewed

Research Article

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

  • Anne-Sophie Archambault,

    Affiliations 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

    ⨯
  • Caroline Turcotte,

    Affiliations 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

    ⨯
  • Cyril Martin,

    Affiliation Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada

    ⨯
  • Julie S. Lefebvre,

    Affiliation Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada

    ⨯
  • Véronique Provost,

    Affiliation Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Québec City, Québec, Canada

    ⨯
  • Michel Laviolette,

    Affiliations 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

    ⨯
  • Nicolas Flamand

    Nicolas.Flamand@criucpq.ulaval.ca

    Affiliations 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

    ⨯

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.

Citation: Archambault A-S, Turcotte C, Martin C, Lefebvre JS, Provost V, Laviolette M, et al. (2017) 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. PLoS ONE 12(1): e0169804. https://doi.org/10.1371/journal.pone.0169804

Editor: Patricia T. Bozza, Fundacao Oswaldo Cruz, BRAZIL

Received: October 7, 2016; Accepted: December 21, 2016; Published: January 9, 2017

Copyright: © 2017 Archambault et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by grants to NF from the Merck Sharpe & Dohme - Faculté de médecine de l'Université Laval, the Natural Sciences and Engineering Research Council of Canada, and the Fonds sur les maladies respiratoires J.-D. Bégin - P.-H. Lavoie. ASA was the recipient of a training award from the Respiratory Health Network of the Fonds de recherche en Santé-Québec and is the recipient of a master degree award from the Fonds de recherche du Québec-Santé. ML and NF are members of the inflammation group of the Respiratory Health Network of the Fonds de recherche en Santé-Québec. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors declare that they have no conflict of interest with any company, notably Merck Sharpe & Dohme. In fact, Merck Sharpe & Dohme gave money to the Université Laval Foundation and had no other involved than providing the funding. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction

Leukotrienes (LT) are inflammatory lipid mediators derived from arachidonic acid. They participate in the inflammatory cascade in numerous conditions, notably asthma, rheumatoid arthritis, allergies and in host defense [1, 2]. They are mainly biosynthesized by leukocytes via the 5-lipoxygenase (5-LO) pathway. With the help of its activating protein, 5-LO metabolizes arachidonic acid into the unstable intermediate LTA4. LTA4 can subsequently be metabolized into LTB4 by the LTA4 hydrolase, or into LTC4 by the LTC4 synthase. Cysteinyl-LTs are well known for their role in asthma and bronchoconstriction, while LTB4 is more involved in leukocyte recruitment and activation. In humans, LTB4 can be further metabolized into 12-oxo-LTB4 by the LTB4 12-hydroxydehydrogenase, or it can be ω-oxidized by the CYP4F3A [3–5]. The latter, which is mainly expressed in neutrophils, catalyzes the formation of 20-OH-LTB4, then 20-COOH-LTB4.

The recognized implication of LTB4 in inflammation makes it an attractive therapeutic target. However, the inhibition of LTB4 biosynthesis showed mixed results in clinical trials, despite promising results in mice models of inflammatory diseases [1, 2]. Among the numerous compounds tested, only the 5-LO inhibitor Zileuton has been approved as a treatment for asthma. Therefore, a better understanding of LTB4 metabolism and its regulation could lead to new therapeutic approaches.

Two recent studies linked the mTOR pathway component p70S6 kinase 1 (p70S6K1) to LTC4 biosynthesis, showing that p70S6K1 could phosphorylate the LTC4 synthase, hence modulating its activity [6, 7]. Herein, we sought to determine whether p70S6K1 could also modulate LTB4 biosynthesis and metabolism. We thus evaluated the impact of two selective p70S6K1 inhibitors, PF-4708671 [8] and LY2584702 [9], on the biosynthesis of LTB4 and its metabolites in human neutrophils.

Materials and Methods

Material

Lymphocyte separation medium, aprotinin, dimethyl sulfoxide (DMSO) and solvents for HPLC and LC/MS were purchased from Thermo Fisher Scientific (Ottawa, Ontario, Canada). Dextran, adenosine deaminase, leupeptin and potassium phosphate were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). HBSS was purchased from Wisent Bioproducts (St-Bruno, Quebec, Canada). 19-OH-prostaglandin (PG) B2, PGB2, PGB2-D4 and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). PF-4708671 was obtained from Abcam (Cambridge, Massachusetts, USA) and LY2584702 from Selleckchem (Houston, Texas, USA). Thapsigargin was obtained from Tocris Bioscience (Ellisville. Missouri, USA). LTB4 was a generous gift from Dr Louis Flamand (Université Laval, Québec City, Canada). Recombinant CYP4F3A and the NADPH regenerating system were purchased from Corning (Corning, New York, USA). Protease and phosphatase inhibitor cocktail tablets were purchased from Roche (Laval, Quebec, Canada). Primary (anti-phospho-S6 #2211 and anti-S6 #2317) and secondary antibodies were obtained from Cell Signaling (Danvers, Massachusetts, USA). The enhanced chemiluminescent (ECL) substrate was obtained from Millipore Canada Ltd (Toronto, Ontario, Canada).

Preparation and utilization of adenosine deaminase

Adenosine deaminase was prepared and utilized exactly as described before [10].

Isolation of human neutrophils and cell stimulations

Human neutrophils were isolated from the peripheral blood of healthy volunteers, without consideration for gender, as described before [11]. For the experiments investigating the impact of p70S6 kinase inhibitors on LTB4 biosynthesis and LTB4 half-life, pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl2) were incubated with PF-4708671, LY2584702 or vehicle (DMSO) for 5 minutes, then stimulated with 100 nM thapsigargin for 10 minutes or 1 μM LTB4 for different times (see Figures). For experiments in which the reversibility of PF-4708671 and 17-ODYA were assessed, pre-warmed human neutrophil suspensions were incubated with PF-4708671, 17-ODYA or vehicle (DMSO) for 5, 15 or 30 minutes. Cells were centrifuged (350 × g) and the pellets were suspended in HBSS-CaCl2 or autologous plasma for 20 minutes. Cells were washed 3 times with warm HBSS-CaCl2 before adding 1 μM LTB4 for 20 minutes.

Analysis of LTB4 and its ω-oxidation products

Incubations were stopped by adding 1 volume of a cold (-30°C) stop solution (MeOH/MeCN, 1/1, v/v) containing 12.5 ng of both 19-OH-PGB2 and PGB2 as internal standards. The samples were placed at -30°C overnight to allow protein denaturation and then centrifuged (1000 × g, 10 minutes, 4°C). The resulting supernatants were analyzed by reversed-phase HPLC using a Shimadzu HPLC System (Shimadzu corporation, Kyoto, Japan) and an on-line extraction procedure based on a method from Borgeat et al [12]. In brief, samples were diluted 1/3 with water, then injected and loaded onto a C8 precolumn (Aquapore Octyl 7 μM, PerkinElmer, Waltham, USA) using water containing 0.01% phosphoric acid during 4.25 minutes. The C8 precolumn was then switched on-line with the HPLC analytical column (Accucore™ C18, 50 × 4.6 mm, 2.6 μm, ThermoFisher Scientific, Ottawa, Canada) and elution was performed using a discontinuous binary gradient with solvent A and solvent B. Solvent A consisted of MeOH/MeCN/H2O at a 44/11/45 ratio (v/v/v) plus 0.01% AcOH and DMSO. Solvent B consisted of MeOH/MeCN/H2O at a 63/32/5 ratio (v/v/v) plus 0.01% AcOH. The gradient was: 0–15% B from 4.25 to 5.25 minutes; 15–70% B from 5.25 to 12 minutes; 70–100% B from 12 to 12.30 minutes and held at 100% during 4 minutes. Column then was re-equilibrated into 15% solvent B during 5 minutes before the next sample was injected. Using this method, the retention times were 5.3 minutes for 19-OH-PGB2, 6.4 minutes for 20-COOH-LTB4, 6.7 minutes for 20-OH-LTB4, 8.9 minutes for PGB2, 9.8 minutes for 6Z-LTB4, 9.9 minutes for 6Z-12epi-LTB4, 10.1 minutes for LTB4, and 12.5 minutes for 5-HETE. Internal standards and LTs were detected by UV at 270 nm while 5-HETE was detected at 235 nm. Leukotrienes represent the sum of LTB4, 20-OH-LTB4 and 20-COOH-LTB4.

In vitro CYP4F3A assay

Human recombinant CYP4F3A (5 pg/ml) in potassium phosphate buffer (100 mM, pH 7.4) containing a NADPH generating system (glucose-6-phosphate, NADP+, glucose-6-phosphate dehydrogenase, MgCl2) was warmed at 37°C then incubated for 5 minutes with inhibitors or vehicle (DMSO). LTB4 (1-20 μM) then was added and reactions were stopped at different times with 5 volumes of a cold stop solution. LTB4 and its ω-oxidation products were quantified by HPLC as described in methods. The initial reaction rate for each LTB4 concentration was determined. The maximal velocity (vmax) and the Michaelis-Menten constant (KM) were calculated for each concentration of PF-4708671 to assess the type of inhibition, using non-linear regression of the Michaelis-Menten graph with the Graphpad Prism 7 Software (GraphPad Software, Inc., La Jolla, California, USA). The Michaelis-Menten graph was also linearized using the Lineweaver-Burk (double reciprocal) plot.

Immunoblot

Pre-warmed neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl2) were stimulated with 100 nM of thapsigargin or N-Formylmethionine-leucyl-phenylalanine (fMLP) for 5 minutes. PF-4708671, LY2584702 or vehicle were added 5 minutes before stimulation. Incubations were stopped using 1 volume of cold (4°C) incubation buffer. The suspensions were centrifuged (350 x g, 5 min, 4°C) and then lysed in a cold (4°C) hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, pH 7.4) containing 0.1% NP-40, protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupetin, 1 mM PMSF, protease inhibitor cocktail tablets), 2 mM diisopropyl fluorophosphate (DFP) and phosSTOP. Cells were vortexed for 15 seconds, then immediately solubilized in electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol, 2% SDS) and boiled for 10 minutes. Proteins were loaded on a 12% polyacrylamide gel for electrophoresis, and transferred onto a PVDF membrane. Membranes were blocked using TBS/Tween buffer containing 5% w/v skim milk and incubated overnight at 4°C with primary antibodies (anti-phospho-S6 #2211 and anti-S6 #2317, Cell Signaling) in TBS/Tween containing 5% skim milk. HRP-linked secondary antibodies and ECL substrate were used for detection.

Quantification of PF-4708671 by LC-MS/MS

Incubations were stopped by adding one volume of cold (-30°C) MeOH + 0.01% acetic acid containing 2 ng of PGB2-D4 as an internal standard. The samples were placed at -30°C overnight to allow protein denaturation and then centrifuged (1000 × g, 10 minutes). The resulting supernatants were collected and diluted with water + 0.01% acetic acid to obtain a final MeOH concentration ≤ 10%. Lipids were extracted from the samples using solid phase extraction cartridges (Strata-X Polymeric Reversed Phase, 60 mg/1ml, Phenomenex). The eluate was dried under a stream of nitrogen and reconstituted in 50 μl of MeOH. 1 μl was injected onto an HPLC column (Kinetex C8, 150 × 2.1 mm, 2.6 μm, Phenomenex) and eluted at a flow rate of 500 μl/min with a linear gradient using 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). The gradient lasted 20 minutes, starting at 10:90 (A:B) a finishing at 90:10 (A:B). The HPLC system was interfaced with the electrospray source of a Shimadzu 8050 triple quadrupole mass spectrometer and mass spectrometric analysis was done in the negative ion mode using multiple reaction monitoring for the specific mass transition m/z 389.10 → 197.95.

Statistical analyses

Data are represented as the mean ± S.D. All calculations were done using the Graphpad Prism 7 Software.

Ethics

This study was approved by the local ethics committee (Comité d’éthique de la recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec) and all subjects signed a consent form.

Results

p70S6 kinase 1 inhibitors do not inhibit the biosynthesis of LTB4 but PF-4708671 prevents further LTB4 metabolism

In the first series of experiments, we investigated whether p70S6K1 inhibitors modulated LTB4 biosynthesis. As shown in Fig 1A, PF-4708671 and LY2584702 at a concentration of 10 μM did not stimulate nor inhibit the thapsigargin-induced LTB4 biosynthesis. At this concentration, both compounds inhibited the phosphorylation of S6 induced by thapsigargin or fMLP (Fig 1B), indicating that they were both efficiently inhibiting the p70S6K1. However, we noticed that 10 μM PF-4708671 prevented the metabolism of LTB4 into 20-OH-LTB4 and 20-COOH-LTB4 in thapsigargin-stimulated neutrophils with an IC50 of ~800 nM (Fig 1C). This led to a decrease in 20-OH- and 20-COOH-LTB4 levels, and an increase in LTB4 levels. In contrast, LY2584702 did not prevent the metabolism of LTB4 into 20-OH- and 20-COOH-LTB4 in thapsigargin-stimulated neutrophils (Fig 1D).

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Fig 1. Impact of the p70S6K1 inhibitors on LTB4 biosynthesis and ω-oxidation in neutrophil suspensions.

A-D) Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl2) were incubated with PF-4708671, LY2584702 or vehicle (DMSO) for 5 minutes, then stimulated with 100 nM thapsigargin for 10 minutes. A,C,D) Samples were processed and analyzed for LTB4 biosynthesis as described in methods. Data are the mean (± S.D) of 5 independent experiments, each performed in duplicate. A) Leukotrienes represent the sum of LTB4, 20-OH-LTB4 and 20-COOH-LTB4. C,D) ω-LTB4 represents the sum of 20-OH-LTB4 and 20-COOH-LTB4. B) Samples were processed and analyzed for S6 and phospho-S6 content as described in methods. Data are from one experiment, representative of three.

https://doi.org/10.1371/journal.pone.0169804.g001

We next determined if PF-4708671 could also inhibit the degradation of exogenously added LTB4 to 20-OH- and 20-COOH-LTB4 by performing kinetic experiments in which neutrophils were incubated with 1 μM LTB4. PF-4708671 increased the half-life of LTB4 by 7.5 fold, from ~20 minutes to ~150 minutes (Fig 2A). In contrast, LY2584702 did not significantly modulate LTB4 half-life (Fig 2B). In comparison, the CYP4F3A inhibitor 17-ODYA, previously shown to inhibit the metabolism of LTB4 into 20-OH-LTB4 in human neutrophils [13], increased LTB4 half-life by 5 fold, from ~10 minutes to ~50 minutes (Fig 2C). In these experiments, neutrophils were treated with PF-4708671 and 17-ODYA during 5 and 30 minutes respectively before the addition of LTB4. This is because PF-4708671 exerted its inhibitory constraint almost instantaneously while the optimal inhibitory effect of 17-ODYA was observed after 30 minutes (Fig 2D). Of note, the conversion of LTB4 into 20-OH- and 20-COOH-LTB4 only occured when neutrophils were present in the incubation media (Fig 2E). Indeed, LTB4 was not transformed into ω-LTB4 in our incubation medium (HBSS) or in a neutrophil supernatant, but was efficiently ω-oxidized in neutrophil suspensions, supporting the fact that the ω-oxidation of LTB4 is an intracellular event.

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Fig 2. Impact of p70S6K1 and CYP4F3A on the half-life of LTB4 in cellulo.

Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl2) were incubated 5 minutes with vehicle (DMSO), A) PF-4708671 (30 μM), B) LY2584702 (10 μM), or 30 minutes with C) 17-ODYA (30 μM), then treated with 1 μM of LTB4 for the indicated times. D) PF-4708671 (30 μM) or 17-ODYA (30 μM) were added to neutrophil suspensions for the indicated times before adding 1 μM LTB4 for 20 minutes. E) LTB4 (1 μM) was added for 20 minutes to either incubation medium, neutrophil supernatants (incubation medium incubated with neutrophils during 30 minutes) or neutrophil suspensions. A-E) Samples then were processed and analyzed for LTB4 biosynthesis as described in methods. ω-LTB4 represents the sum of 20-OH-LTB4 and 20-COOH-LTB4. Data are the mean (± S.D) of 4-5 independent experiments, each performed in duplicate.

https://doi.org/10.1371/journal.pone.0169804.g002

PF-4708671 is a mixed inhibitor of CYP4F3A

The distinct inhibitory profiles of PF-4708671 and LY2584702 on LTB4 metabolism raised the possibility that PF-4708671 was a CYP4F3A inhibitor. We thus tested this hypothesis by comparing the effect of p70S6K1 inhibitors with the CYP4F3A inhibitor 17-ODYA on human recombinant CYP4F3A activity. Human recombinant CYP4F3A was incubated with increasing concentrations of PF-4708671 or 17-ODYA for 5 and 30 minutes respectively, before the addition of 1 μM of LTB4 for 1 minute. PF-4708671 induced a concentration-dependent inhibition of ω-oxidation product formation (IC50 ~750 nM) while LY2584702 poorly affected this enzymatic conversion (Fig 3A). In other experiments, CYP4F3A was treated with increasing concentrations of PF-4708671 before the addition of various LTB4 concentrations. We then calculated the maximal rate of the reaction (vmax) and the Michaelis-Menten constant (KM) during the steady-state of the reaction using the non-linear regression of the Michaelis-Menten graph. The representation of the data using either the Michaelis-Menten graph (Fig 3C) or the Lineweaver-Burk plot (Fig 3D) are consistent with a model of mixed inhibition. This mixed inhibition indicated that PF-4708671 was perhaps a substrate of CYP4F3A. However, LC-MS/MS analyses of PF-4708671 levels indicated that the compound remained stable in our neutrophil suspensions and in our enzymatic assay with recombinant CYP4F3A for up to 2 hours (data not shown).

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Fig 3. Impact of PF-4708671 on human recombinant CYP4F3A activity.

A) Pre-warmed human recombinant CYP4F3A in phosphate buffer containing a NADPH regenerating system was incubated with increasing concentrations of PF-4708671 for 5 minutes, with LY2584702 (10-5M) for 5 minutes or with 17-ODYA (10-5M) for 30 minutes before the addition of 1 μM LTB4 for 1 minute. B) Representative time curves of the ω-oxidation of LTB4 par CYP4F3A. Initial rates were calculated by linear regession. C) Michaelis-Menten kinetics of the inhibition of CYP4F3A by PF-4708671. Human recombinant CYP4F3A was incubated with increasing concentrations of PF-4708671 before the addition of LTB4 at different concentrations. LTB4, and ω-LTB4 (20-OH-LTB4 and 20-COOH-LTB4) were quantified by HPLC as described in methods and represent the addition of 20-OH-LTB4 and 20-COOH-LTB4. Data are shown as the mean (± S.D) of 8 independent experiments. D) Data from the Michaelis-Menten graph were transformed using the Lineweaver-Burk plot (double reciprocal plot). E) Legend for C and D.

https://doi.org/10.1371/journal.pone.0169804.g003

The inhibitory effect of PF-4708671 on LTB4 ω-oxidation is reversible

Finally, we assessed if the inhibitory effect of PF-4708671 was reversible by comparing it with the irreversible CYP4F3A inhibitor 17-ODYA [13]. In these experiments, neutrophils were incubated with PF-4708671 for 5 minutes or 17-ODYA for 30 minutes (optimal incubation times) or with the inhibitors for 15 minutes (comparable incubation time). Then, neutrophils were either washed with HBSS-CaCl2, washed with autologous plasma or not washed at all, before treatement with either 100 nM thapsigargin or 1 μM LTB4. In absence of washing, both PF-4708671 and 17-ODYA inhibited the metabolism of LTB4 into 20-OH- and 20-COOH-LTB4 (Fig 4). Washing the PF-4708671-treated neutrophils with either HBSS-CaCl2 or autologous plasma restored the ability of neutrophils to metabolize endogenously formed and exogenously added LTB4 into 20-OH- and 20-COOH-LTB4 (Fig 4A–4C). In contrast, 17-ODYA-treated neutrophils that were washed with either HBSS-CaCl2 or autologous plasma remained incapable of metabolizing endogenously biosynthesized or exogenously added LTB4 into 20-OH- or 20-COOH-LTB4 (Fig 4D–4F). Furtermore, similar results were obtained whether we used the optimal incubation time for each inhibitor or the comparable incubation time (5 and 30 minutes for PF-4708671 and 17-ODYA vs. 15 minutes for each inhibitor). These experiments indicate that in contrast to the irreversible inhibitor 17-ODYA, the inhibitory constraint of PF-4708671 is easily removable and that the latter is a reversible inhibitor of the CYP4F3A enzyme.

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Fig 4. Removal of the inhibitory constraint exerted by CYP4F3A inhibitors on LTB4 ω-oxidation in neutrophils.

Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl2) were incubated with A,B) 30 μM PF-4708671 or vehicle for 5 minutes, C) 30 μM PF-4708671 for 15 minutes, D,E) 30 μM 17-ODYA for 30 minutes, or F) 30 μM 17-ODYA for 15 minutes. Neutrophils were washed (or not) with autologous plasma or HBSS-CaCl2 as described in methods. A,D) 100 nM thapsigargin or B,C,E,F) 1 μM LTB4 were then added for 10 and 20 minutes, respectively. Samples then were processed and analyzed for 20-OH-LTB4 and 20-COOH-LTB4 as described in methods. Data are the mean (± S.D) of 4 independent experiments, each performed in duplicate.

https://doi.org/10.1371/journal.pone.0169804.g004

Discussion

Two recent studies have linked the mTOR pathway component p70S6K1 to LTC4 synthase function, providing a possible new way of regulating cysteinyl-LT biosynthesis [6, 7]. Thus, we wondered if p70S6K1 could play a similar role in the regulation of LTB4 synthesis. In that regard, our data show that 1) the thapsigargin-induced LTB4 biosynthesis is not inhibited by p70S6K1 inhibitors; 2) PF-4708671, but not LY2584702, inhibits the metabolism of LTB4 into 20-OH- and 20-COOH-LTB4 in a concentration-dependant manner; 3) PF-4708671, but not LY2584702, inhibits human recombinant CYP4F3A; 4) PF-4708671 is a reversible inhibitor of CYP4F3A; and 5) PF-4708671 is a mixed inhibitor of CYP4F3A.

In this study, we aimed at documenting the inhibitory effect of p70S6K1 inhibitors on the ability of human neutrophils to metabolize LTB4 into 20-OH- and 20-COOH-LTB4. For that reason, we utilized an experimental model in which the priming of the arachidonic acid cascade and the 5-LO pathway were not involved, i.e. thapsigargin-stimulated neutrophils. In that experimental model, we could show that the ability of neutrophils to biosynthesize LTB4, which includes the sum of LTB4, 20-OH-LTB4, and 20-COOH-LTB4, was unchanged. This indicates that at the concentration utilized, the p70S6K1 inhibitors PF-4708671 and LY2584702 did not inhibit the enzymes involved in LTB4 biosynthesis in human neutrophils, notably cPLA2α, 5-LO, 5-LO-activating protein and LTA4 hydrolase [14–17]. However, it remains possible that the p70S6K1 inhibitors used in this study might impact LTB4 biosynthesis in human neutrophils when other signaling mechanisms are involved, notably those linked to the priming of LTB4 biosynthesis such as cytokines and TLR activation [18, 19], raising the possibility that PF-4708671, and possibly LY2584702, modulate LT biosynthetic pathways through multiple mechanisms of action.

We found that PF-4708671, but not LY2584702, inhibited the metabolism of LTB4 into 20-OH- and 20-COOH-LTB4 in a concentration-dependant manner. In that regard, PF-4708671 was more potent than 17-ODYA, a recognized CYP4F3A inhibitor [13]. Moreover, the effect of PF-4708671 was more pronounced that of 17-ODYA. While PF-4708671 increased the half-life of LTB4 in our human neutrophil suspensions by 7.5 fold, 17-ODYA increased it by 5 fold. This indicates that PF-4708671 might be a promising tool to develop specific and potent inhibitors of the CYP4F3A enzyme.

Given that the Michaelis-Menten and the Lineweaver-Burk plots indicated that PF-4708671 was a mixed inhibitor, we thought that perhaps PF-4708671 was a CYP4F3A substrate and was metabolized to some extent by the enzyme. However, this hypothesis was proven incorrect, as PF-4708671 was stable for at least 2 hours in the presence of either recombinant CYP4F3A or human neutrophils. The mixed inhibition we observed also raised the possibility that a contaminant in our commercial PF-4708671 preparation might also inhibit CYP4F3A. Although we cannot infirm that possibility, we tested three different batches of PF-4708671 which all yielded the same results, indicating that the effects we are documenting are very unlikely to be caused by a compound that is present in trace amounts.

This is not the first study to underscore a non-specific effect of PF-4708671. Another group reported an off-target effect of PF-4708671 in immortalized mouse fibroblasts, showing that PF-4708671 activates AMPK and inhibits the mitochondrial respiratory chain complex I, independently of p70S6K1 [20]. Moreover, PF-4708671 is used as a tool to study the mTOR pathway in various in vivo and in vitro studies, mainly in models of type 2 diabetes and cancer [21–33]. In light of our findings, it cannot be ruled out that some of the previously reported effects of PF-4708671 are caused by its lack of specificity and possibly increased LTB4 levels.

It was previously reported that LTB4 levels are increased in white adipose tissue, liver and muscles of mice fed with an high fat diet [34, 35]. Furthermore, mice lacking LTB4 receptor 1 are less susceptible to diet-induced insulin resistance [36, 37]. Therefore, using PF-4708671 in models of type 2 diabetes could increase LTB4 levels and possibly lead to an increased inflammation. However, in rodents, the functional orthologue of CYP4F3A is CYP4F18, which transforms LTB4 into 18-OH-LTB4 instead of 20-OH-LTB4 [38, 39]. While the activity of CYP4F18 towards LTB4 has been characterized, it is still unknown whether PF-4708671 exerts an inhibitory effect on CYP4F18 as well.

In conclusion, we demonstrate that PF-4708671 is a reversible CYP4F3A inhibitor preventing the metabolism of LTB4 into 20-OH- and 20-COOH-LTB4. In addition to characterizing a new compound that induces a sustained elevation in LTB4 levels, our data shed some light on the non-specific effects of a widely used p70S6K1 inhibitor. Given that it is more potent than the only currently available CYP4F3A inhibitor, PF-4708671 might be an helpful tool for the development of potent CYP4F3A inhibitors to study the regulation of LTB4 metabolism and its impact in inflammation.

Acknowledgments

ML and NF are members of the inflammation group of the Respiratory Health Network of the Fonds de recherche du Québec-Santé.

Author Contributions

  1. Conceptualization: ASA JSL VP ML NF.
  2. Formal analysis: ASA NF.
  3. Funding acquisition: ML NF.
  4. Investigation: ASA CM NF.
  5. Methodology: ASA CM NF.
  6. Resources: ML NF.
  7. Supervision: ML NF.
  8. Visualization: ASA NF.
  9. Writing – original draft: ASA CT NF.
  10. Writing – review & editing: ASA CT CM JSL VP ML NF.

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