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

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.


Introduction
Leukotrienes (LT) are inflammatory lipid mediators derived from arachidonic acid. They participate in the inflammatory cascade in numerous conditions, notably asthma, rheumatoid a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 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 LTA 4 . LTA 4 can subsequently be metabolized into LTB 4 by the LTA 4 hydrolase, or into LTC 4 by the LTC 4 synthase. Cysteinyl-LTs are well known for their role in asthma and bronchoconstriction, while LTB 4 is more involved in leukocyte recruitment and activation. In humans, LTB 4 can be further metabolized into 12-oxo-LTB 4 by the LTB 4 12-hydroxydehydrogenase, or it can be ω-oxidized by the CYP4F3A [3][4][5]. The latter, which is mainly expressed in neutrophils, catalyzes the formation of 20-OH-LTB 4 , then 20-COOH-LTB 4 .
The recognized implication of LTB 4 in inflammation makes it an attractive therapeutic target. However, the inhibition of LTB 4 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 LTB 4 metabolism and its regulation could lead to new therapeutic approaches.
Two recent studies linked the mTOR pathway component p70S6 kinase 1 (p70S6K1) to LTC 4 biosynthesis, showing that p70S6K1 could phosphorylate the LTC 4 synthase, hence modulating its activity [6,7]. Herein, we sought to determine whether p70S6K1 could also modulate LTB 4 biosynthesis and metabolism. We thus evaluated the impact of two selective p70S6K1 inhibitors, PF-4708671 [8] and LY2584702 [9], on the biosynthesis of LTB 4 and its metabolites in human neutrophils.

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 LTB 4 biosynthesis and LTB 4 half-life, pre-warmed human neutrophil suspensions (37˚C, 5 million cells/ml in HBSS containing 1.6 mM CaCl 2 ) were incubated with PF-4708671, LY2584702 or vehicle (DMSO) for 5 minutes, then stimulated with 100 nM thapsigargin for 10 minutes or 1 μM LTB 4 for different times (see Figures). For experiments in which the reversibility of PF-4708671 and 17-ODYA were assessed, prewarmed 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-CaCl 2 or autologous plasma for 20 minutes. Cells were washed 3 times with warm HBSS-CaCl 2 before adding 1 μM LTB 4 for 20 minutes.

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 PGB 2 -D 4 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.

p70S6 kinase 1 inhibitors do not inhibit the biosynthesis of LTB 4 but PF-4708671 prevents further LTB 4 metabolism
In the first series of experiments, we investigated whether p70S6K1 inhibitors modulated LTB 4 biosynthesis. As shown in Fig 1A, PF-4708671 and LY2584702 at a concentration of 10 μM did not stimulate nor inhibit the thapsigargin-induced LTB 4 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 LTB 4 into 20-OH-LTB 4 and 20-COOH-LTB 4 in thapsigargin-stimulated neutrophils with an IC 50 of~800 nM (Fig 1C). This led to a decrease in 20-OH-and 20-COOH-LTB 4 levels, and an increase in LTB 4 levels. In contrast, LY2584702 did not prevent the metabolism of LTB 4 into 20-OH-and 20-COOH-LTB 4 in thapsigargin-stimulated neutrophils (Fig 1D).
We next determined if PF-4708671 could also inhibit the degradation of exogenously added LTB 4 to 20-OH-and 20-COOH-LTB 4 by performing kinetic experiments in which neutrophils were incubated with 1 μM LTB 4 . PF-4708671 increased the half-life of LTB 4 by 7.5 fold, from~20 minutes to~150 minutes (Fig 2A). In contrast, LY2584702 did not significantly modulate LTB 4 half-life (Fig 2B). In comparison, the CYP4F3A inhibitor 17-ODYA, previously shown to inhibit the metabolism of LTB 4 into 20-OH-LTB 4 in human neutrophils [13], increased LTB 4 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 LTB 4 . 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 LTB 4 into 20-OH-and 20-COOH-LTB 4 only occured when neutrophils were present in the incubation media ( Fig 2E). Indeed, LTB 4 was not transformed into ω-LTB 4 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 LTB 4 is an intracellular event.

PF-4708671 is a mixed inhibitor of CYP4F3A
The distinct inhibitory profiles of PF-4708671 and LY2584702 on LTB 4 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 LTB 4 for 1 minute. PF-4708671 induced a concentration-dependent inhibition of ω-oxidation product formation (IC 50~7 50 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 LTB 4 concentrations. We then calculated the maximal rate of the reaction (v max ) and the Michaelis-Menten constant (K M ) 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).

The inhibitory effect of PF-4708671 on LTB 4 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-CaCl 2 , washed with autologous plasma or not washed at all, before treatement with either 100 nM thapsigargin or 1 μM LTB 4 . In absence of washing, both PF-4708671 and 17-ODYA inhibited the metabolism of LTB 4 into 20-OH-and 20-COOH-LTB 4 (Fig 4). Washing the PF-4708671-treated neutrophils with either HBSS-CaCl 2 or autologous plasma restored the ability of neutrophils to metabolize endogenously formed and exogenously added LTB 4 into 20-OH-and 20-COOH-LTB 4 (Fig 4A-4C). In contrast, 17-ODYA-treated neutrophils that were washed with either HBSS-CaCl 2 or autologous plasma remained incapable of metabolizing endogenously biosynthesized or exogenously added LTB 4 into 20-OH-or 20-COOH-LTB 4 (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.

Discussion
Two recent studies have linked the mTOR pathway component p70S6K1 to LTC 4 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 LTB 4 synthesis. In that regard, our data show that 1) the thapsigargin-induced LTB 4 biosynthesis is not inhibited by p70S6K1 inhibitors; 2) PF-4708671, but not LY2584702, inhibits the metabolism of LTB 4 into 20-OH-and 20-COOH-LTB 4 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 LTB 4 into 20-OH-and 20-COOH-LTB 4 . 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 LTB 4 , which includes the sum of LTB 4 , 20-OH-LTB 4 , and 20-COOH-LTB 4 , was unchanged. This indicates that at the concentration utilized, the p70S6K1 inhibitors PF-4708671 and LY2584702 did not inhibit the enzymes involved in LTB 4 biosynthesis in human neutrophils, notably cPLA 2 α, 5-LO, 5-LO-activating protein and LTA 4 hydrolase [14][15][16][17]. However, it remains possible that the p70S6K1 inhibitors used in this study might impact LTB 4 biosynthesis in human neutrophils when other signaling mechanisms are involved, notably those linked to the priming of LTB 4 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 LTB 4 into 20-OH-and 20-COOH-LTB 4 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 LTB 4 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][22][23][24][25][26][27][28][29][30][31][32][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 LTB 4 levels.
It was previously reported that LTB 4 levels are increased in white adipose tissue, liver and muscles of mice fed with an high fat diet [34,35]. Furthermore, mice lacking LTB 4 receptor 1 are less susceptible to diet-induced insulin resistance [36,37]. Therefore, using PF-4708671 in models of type 2 diabetes could increase LTB 4 levels and possibly lead to an increased inflammation. However, in rodents, the functional orthologue of CYP4F3A is CYP4F18, which transforms LTB 4 into 18-OH-LTB 4 instead of 20-OH-LTB 4 [38,39]. While the activity of CYP4F18 towards LTB 4 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 LTB 4 into 20-OH-and 20-COOH-LTB 4 . In addition to characterizing a new compound that induces a sustained elevation in LTB 4 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 LTB 4 metabolism and its impact in inflammation.